1 Department of Internal Medicine, Section of Applied and Clinical Biochemistry, University of Perugia, and 2 NephrologyDialysis Unit, R. Silvestrini Hospital, Perugia, Italy
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Abstract |
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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 <CRF <HD). Diabetic patients on SHD showed similar levels of glycation indexes as non-diabetic patients, except for the early product furosine that was notably higher. The shift from SHD to DHD was effective in lowering the concentration of all the glycation parameters measured, both in non-diabetic and diabetic patients. In the total HD population, LMM-AGEs (MM range of approx. 1.56.0 kDa) detected at 385 nm emission was lowered by 56% (P<0.001) and LMM-AGEs detected at 440 nm emission and furosine decreased by 23 and 19%, (P0.001 and <0.01, respectively). All these three classes of compounds reached concentrations comparable with those observed in the CRF patients, even if remaining above the control range. The levels of both free and protein-bound pentosidine after DHD decreased by 34% (P<0.001) and 22% (P
0.05), respectively. The return for 3 months to SHD in four non-diabetic DHD patients led to a trend toward an increase in all five glycation parameters.
Conclusions. This study demonstrates for the first time that a DHD regimen can effectively lower the mean levels of glycation-related substances observed in SHD. Therefore, DHD can provide a better control of AGE produced in ESRD. This could result in a lower incidence of long-term effects of AGE accumulation in HD.
Keywords: AGEs; furosine; daily haemodialysis; pentosidine; protein glycation; uraemic toxins
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Introduction |
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Under normal conditions, the protein turnover in tissue and plasma maintains protein glycation products at trace levels. Significant in vivo accumulation of AGE-modified proteins has been observed during the ageing process, and, at great extent, also in diabetic patients [3]. More recently, increased levels of AGE proteins and AGE peptides have been reported also in the plasma and tissues of end-stage renal disease (ESRD) patients on haemodialysis (HD), independently from the presence of hyperglycaemic conditions [46].
AGE peptides deriving from the catabolism of glycated proteins increase dramatically in plasma of patients undergoing HD, due to the anomaly in the chemistry of the uraemic environment, impaired renal function, and inadequate removal by current dialysis treatments [7]. In uraemic patients, plasma AGE peptides that retain chemically active products of glycation tend to react with target proteins in plasma and in the vessel wall, particularly collagen; in plasma, the main substrates of glycation are albumin [8], ß2 microglobulin [9], and ancillary proteins of LDL [10]. Therefore, the high glycation rate in chronic uraemia appears to be due to an abnormal synthesis of so-called second generation AGEs [11], which might contribute to the onset and progression of severe co-morbidity in ESRD, such as atherosclerosis and amyloidosis.
Moreover, the accumulation of AGEs observed in uraemia could be caused directly by reactive carbonyls, which react with the amino groups of proteins. These toxins, which are related (or not) to glucose chemistry in uraemia, might be produced also as a consequence of oxidative stress conditions [12].
Early studies have demonstrated that even the most recent dialysis approaches are largely inadequate to reconduce AGE levels steadily close to normal, as renal transplant does [7]. Different types of dialysers, and particularly the high flux ones, have been shown to markedly reduce AGEs during each treatment session but the levels of these compounds returned in a few hours to the pre-treatment amount [7]. Therefore, the features of partial and discontinuous correction of AGE levels of the current HD procedures leaves chronic HD patients exposed for a long time to high levels of these toxins.
An unexplored strategy of improving dialysis efficacy to correct glycation product formation and/or accumulation might be to increase the HD frequency. In order to verify this possibility, we compared in a prospective longitudinal study, the effect on AGE levels of a daily (2 h, 6 times/week) HD schedule (DHD), with that of a standard (4 h, 3 times/week) HD schedule (SHD).
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Subjects and methods |
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At the enrolment, all the patients had a residual creatinine clearance <1 ml/min, the mean dialysis age of the SHD group was 881±358 days and all of the patients underwent bicarbonate dialysis for at least 6 months. Patients were treated with low-flux dialysers (low-flux polysulfone, n=14; cellulose acetate, n=9; cuprammonium rayon, n=9). Throughout the study, the same filter, blood flow, and medications were maintained compatible with the ethical guidelines provided by the Ethics Committee of the R. Silvestrini Hospital. Exclusion criteria included: age >80 or <25 years, severe malnutrition, and malignancies.
Eleven apparently healthy subjects (5 females: age=57±7.5 years; 6 males: age=55±6.4 years) with no clinical history or signs of renal disease were considered as a control group. In accordance with guidelines provided by the Institutional Ethics Committee of the R. Silvestrini Hospital, verbal consent was obtained from all the patients and normal subjects.
Blood sampling was performed before the beginning of the period on DHD and after 6 months of DHD. Blood samples were collected from the arterial line at the start of the mid-week dialysis session. Plasma samples were analysed immediately or stored at -35°C until use.
Blood biochemistry and Kt/V were monitored monthly; Kt/V was calculated with the single pool method.
Analytical procedures
Clinical biochemistry
Creatinine was determined in plasma by Jaffe's assay and plasma proteins by the biuret reaction on a Hitachi 717 analyser (Boeringher Mannheim, Germany).
Protein glycation status
The following glycation parameters were determined with a method previously published [13]: furosine (expressed as pmol/mg protein), free and protein-bound pentosidine (expressed as pmol/ml plasma and pmol/mg protein, respectively), and two classes of LMM-AGE peptides (MM range of approx. 1.56.0 kDa) [7], detected fluorimetrically at Ex/Em 370/440 and 335/385 nm, and calculated as arbitrary units (AU)/ml plasma. Plasma peptides showing a maximum fluorescence at 385 nm contain the glycoxidation product pentosidine, while those fluorescing at 440 nm are considered to contain other epitopes, such as the advanced protein glycation products 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (23). Even if this analysis is not specific for individual peptides, it gives a generic information about the accumulation of different types of AGEs and the effect of dialysis on their levels [13]. Moreover, this peptide analysis is quite sensitive, particularly in the case of the pentosidine that is determined in the same chromatogram also as free form (Figure 1). Furosine is an early glycation index, while pentosidine reflects the real extent of glycoxidation of plasma proteins. LMM-AGEs as well as free pentosidine have been considered to monitor the whole metabolism of major AGEs [4,7,13].
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Statistics
The results are expressed as means±1 standard deviation (SD). The t-test for paired data and the one-way ANOVA test were used. Probability values lower than 5% were accepted, while values of 1% or lower were considered as highly significant.
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Results |
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Figure 2 shows in detail the glycation parameters assessed in healthy controls, in CRF patients, and in non-diabetic and diabetic uraemic patients on HD studied during the step in SHD and after 6 months of DHD. Mean values, percentage decrease after DHD, and statistics are summarized in Table 1
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The protein-bound pentosidine (Figure 2B), a direct marker of glycoxidation processes on plasma and tissue proteins, was present in normal subjects at levels of 1.4±0.3 pmol/mg proteins. In SHD patients, a dramatic increase (approx. 20-fold) of this index was observed (28.3±9.7 pmol/mg protein). After DHD, the mean concentration of pentosidine bound to plasma proteins decreased to 22.0±7.7 pmol/mg protein (P<0.05 vs SHD) but it was still two-fold higher than in CRF patients (9.8±2.5 pmol/mg protein).
Free pentosidine (Figure 2C) was undetectable in the plasma of subjects with normal renal function. The levels of this compound increased to 46.8±19.4 pmol/ml in CRF patients and to 117.3±22.2 pmol/ml in SHD patients. The shift from SHD to DHD lowered the levels of free pentosidine to a mean value of 77.0±16.6 pmol/ml (P<0.001).
The LMM-AGE peptides (Figure 2D) determined by fluorimetric detection at 440 nm (AGEs370/440) were present in control plasma at a concentration of 94.1±8.8 AU/ml. This level is about 4 times and more than 5 times lower than that found in CRF patients (413.9±155.3 AU/ml, P<0.001) and SHD (576.8±141.6 AU/ml, P<0.001), respectively. After DHD, the mean concentration of LMM-AGEs (441.6±113.6 AU/ml) decreased significantly (P<0.001 vs SHD), reaching the same mean level observed in the CRF group.
The LMM-AGE peptides (Figure 2E) detected at 385 nm (AGEs335/385) and, thus, containing pentosidine-like epitopes were significantly lowered after the step on DHD compared with that on SHD (820.8±429.1 and 1859.9±726.0 AU/ml, respectively; P<0.001). It is noteworthy that the treatment for 6 months with the DHD modality gave values lower than those in the CRF group (1032±432.5 AU/ml, P=not significant (NS)). However, the concentration of these compounds still remained several fold higher than normal (30.5±17.4 AU/ml).
The return for 3 months to SHD in 4 non-diabetic DHD patients led to a trend toward an increase in all the glycation parameters and particularly of protein-bound pentosidine.
As shown in Figure 2, in diabetic patients all the glycation parameters were significantly decreased after the treatment with the DHD schedule. The same values of probability than in the non-diabetic sub-group were observed (P<0.05 or higher).
The regression analysis between different glycation parameters in SHD revealed a positive correlation for all the parameters studied (data not shown). This correlation reached the highest statistical significance when the two classes of AGE peptides were compared (r2=0.759, P<0.0001); a remarkable positive correlation was also found between AGE peptides and the levels of the protein-bound pentosidine (r2=0.575 for AGEs355/385 and r2=0.495 for AGEs370/440, both with P<0.0001).
The data observed in SHD correlated with those in DHD, particularly so for pentosidine (r2=0.847, P<0.0001), furosine (r2=0.445, P<0.005), and AGEs370/440 (r2=0.578, P=0.004), while no correlation was observed when AGEs335/385 and free pentosidine were considered (r2=0.098 and 0.179, respectively; both with P=NS). Moreover, a significant positive correlation was observed between the levels of all the glycation parameters, including furosine, observed in SHD and the change of their concentration after the DHD step (data not shown).
Concerning the dialysis dose, the shift from SHD to DHD did not change the Kt/V value significantly (SHD=3.98±0.63, DHD=4.33±0.74, P=NS). No correlation was found between any of the glycation parameters and the Kt/V values. The different types of dialysis membranes did not influence the effect of the DHD on glycation parameters (data not shown).
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Discussion |
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The biochemical and physiopathological mechanisms concerning the AGE accumulation on plasma proteins and extracellular matrix of ESRD patients, particularly when on HD, have not been fully elucidated yet. However, their accumulation is believed to be due to an abnormal synthesis of the so-called second generation AGEs [11], and to oxidative and carbonyl stress [12]. So far, these processes cannot be prevented efficiently, and for this reason one of the most important ways of limiting glycoxidation processes in uraemic patients might be to remove, as far as possible, circulating LMM-AGE peptides and toxins sustaining glycation and glycoxidation of plasma and tissue proteins.
In HD patients, high-flux dialysers can, at least in part, provide a lowering of plasma LMM-AGEs [7,15]. We recently observed that ultra high-flux dialysers (also called protein-leaking dialysers) compared with standard low- and high-flux dialysers can further lower the levels of some classes of glycation products, including LMM-AGEs and free and protein-bound pentosidine measured before dialysis [16,17].
In addition to the use of high-flux dialysers, the dialysis frequency might influence AGE levels removing glycation precursors and providing an overall better correction of the uraemic toxicity. In order to verify this hypothesis, in the present study we prospectively compared the effect of SHD and DHD on some glycation indices that have been measured with an HPLC method set up in our laboratory [13].
The results show that with the progression of renal failure and the entering on a dialysis programme, the accumulation of AGE peptides in plasma and the related protein damage steadily worsen, and this might lead ultimately to an increased tissue deposition of AGEs [7]. Intriguingly, the DHD rhythm compared to the SHD one was observed to lower significantly the levels of all the classes of glycation parameters investigated, in a way proportional to their starting concentrations. An hypothesis to explain the mechanism by which DHD compared with SHD could provide a better efficacy in steadily lowering AGEs levels might be that of providing a more continuous (or frequent) and smooth clearance of glycation precursors and LMM-AGEs, which combined with an overall better correction of the uraemic environment as known for blood pH and toxin peaks [1821], might contribute to positively regulate the production and catabolism of AGEs. Accordingly, some of us demonstrated previously that DHD may correct, at least in part, the expression of non-specific indicators of the uraemic toxicity and oxidant stress such as the induction of the erythrocyte form of the detoxifying enzyme glutathione S-transferase [22].
Moreover, some studies have pointed out the role of DHD in improving the clinical outcome of HD patients, particularly providing conditions for a good cardiovascular compliance [1820,23,24].
In this study, in agreement with other findings in the literature [4], we showed that LMM-AGE peptides (MM <10 kDa) remarkably accumulate in the plasma of CRF patients not yet on dialysis and to a much greater extent in both non-diabetic and diabetic patients on HD. Moreover, we here investigated for the first time LMM-AGE accumulation in chronic uraemia by HPLC determination at two fluorescence emission values (385 and 440 nm). While the recording of peptide fluorescence at 385 nm represents a quantitative measure of the glycoxidation product pentosidine, the fluorescence at 440 nm emission can be used to measure advanced protein glycation products, such as 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole [25]. Both of these heterogeneous classes of peptides are increased significantly in CRF and HD patients. Interestingly, among the parameters investigated in this study, the greatest reduction by shifting patients from SHD to DHD concerned the levels of LMM-AGEs showing the typical fluorescence of the glycoxidation end-product pentosidine (i.e. 385 nm emission) and the levels of the free pentosidine, whereas a lesser decrease was observed in LMM-AGEs with fluorescence at 440 nm emission. Thus, one might speculate on a possible effect of the DHD, particularly in lowering glycoxidation reactions, which could be in some extent related with oxidative stress and carbonyl accumulation in dialysis patients. However, notable was the observation that during the DHD step both classes of LMM-AGEs were lowered down to the levels found in the CRF group or below, as in the case of the AGE peptides measured at 385 nm.
The amount of furosine was increased remarkably both in CRF and HD patients, also in the absence of hyperglycaemic conditions. These results are in agreement with recent observations in HD and CAPD patients [26]. In non-diabetic uraemic patients, increased levels of furosine could be explained mainly by a direct implication of factors depending on the uraemic toxicity and dialysis treatment, such as a lowering of plasma pH or conditions of hyperphosphataemia, which may promote Schiff adduct synthesis [2] and catalyse the formation of the Amadori product [27], respectively. Both these possible determining factors, and particularly blood pH, may be better controlled by DHD [18,19,21,22]. According with these observations, compared with SHD, DHD induced a pronounced decrease in the levels of furosine both in diabetic and non-diabetic patients. In the latter case, furosine returned to the levels observed in CRF patients.
Both the free and protein-bound forms of the glycoxidation end-product, pentosidine, strongly accumulated particularly during the SHD step. This dramatic accumulation of pentosidine was observed to be out of proportion with respect to that of the Amadori product. This disproportion might be explained by factors known to sustain protein glycoxidation and, thus, pentosidine formation in uraemic patients, such as the accumulation of reactive compounds arising from the oxidative stress and/or other biochemical processes leading to reactive carbonyl accumulation [12].
Early studies suggested that the modification of plasma proteins by means of LMM-AGE peptides could play a role in this context, inducing an increased production of second generation AGEs [11,28]. This evidence and its possible biological implications need to be confirmed by further investigation. In this context, it is important to emphasize that the acid hydrolysis of AGE peptides in plasma ultrafiltrate does not release an appreciable amount of pentosidine, demonstrating that only a negligible amount of the total circulating pentosidine (less than 1%) is linked to peptides with a MM <10 kDa. This aspect might help to further improve dialysis strategies that other than, or in combination with, DHD are aimed at removing or slowing down the formation rate of AGEs. These strategies may include the use of high-flux dialysers [7,15,16] or highly biocompatible dialysis membranes and antioxidants [29].
In this study, both dialysis membrane and dialysate composition were maintained throughout the study. Changes in these parameters cannot, therefore, account for the observed results. As regards the role of dialysis dose, one can expect only a marginal influence on the parameters investigated due to the fact that Kt/V was increased by less than 8% after switching from SHD to DHD (P=NS).
The specific role of DHD in comparison with SHD in lowering the levels of these compounds was confirmed in a third step of the prospective study, in which 4 patients on DHD were returned to SHD. Even though the number of subjects studied is low and they were studied after a short time from the return to the SHD schedule (3 months), the tendency of all the glycation parameters to increase was observed. This was particularly obvious in the case of the protein-bound fraction of pentosidine, which thus could be considered for a role as sensitive index of glycoxidation reactions in HD patients and as possible precursor of AGE peptides containing pentosidine and free pentosidine.
The effect of DHD in lowering the levels of these glyc(oxid)ation products appears to be dependent on a more frequent dialysis schedule than that of the SHD and could be centred on the more frequent clearance of AGE precursors, such as reactive carbonyls and LMM-AGEs. This possibility, now under further investigation in our laboratory, is in agreement with the observation that the highest rate of removal of LMM glycation products, similarly to other small toxins such as urea, is obtained during the first 2 h of HD treatment [7]. This means that, even maintaining the same total time of dialysis per week (i.e. 2 h, 6 times vs 4 h, 3 times), with a DHD protocol the frequency of the HD sessions effective in eliminating these classes of AGEs and their precursors is doubled, ultimately improving the global efficacy of their removal.
In conclusion, non-diabetic ESRD patients not yet included in a dialysis programme show a remarkable accumulation of both early and advanced glycation products as compared with normal subjects. Glycation indices are worsened by chronic HD. Compared to normoglycaemic subjects, diabetic patients on SHD show similar levels of glycoxidation parameters and significantly higher levels of the early glycation product furosine. The shift from a SHD to a DHD schedule is effective in lowering the levels of all the glycation products, which even if remaining far above the normal interval in the case of furosine and LMM-AGE peptides, were observed to return to values found in CRF patients not yet on HD. Even if the design of this study can lead only to the conclusion that DHD may lower the peaks of concentration of some glycation molecules, it is important to consider that it provides a direct proof of the effect of this dialysis modality in reducing, at least in a window of time, the concentration of these substances. Thus, it can be proposed that DHD may remove AGEs or slow down the progression of glycation processes that can contribute significantly to uraemic toxicity. These effects are comparable with those of high-flux dialysers [7,15,16] and, thus, a daily schedule with high-flux membranes should be considered for a combined approach aimed to increase the efficacy of dialysis in correcting AGEs in a wide molecular weight range. Further studies are in progress to better elucidate this aspect.
Therefore, this study demonstrates for the first time that DHD compared with SHD can significantly, although partially, correct the levels of some classes of AGEs. On the basis of evidence that AGE accumulation may have several adverse biological roles [2,811], our findings suggest a favourable impact of DHD in lowering the risk of AGE-related long-term complications in HD patients, such as amyloidosis and atherosclerotic cardiovascular disease.
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Acknowledgments |
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Notes |
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References |
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