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NDT Advance Access published online on November 25, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn634
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


AGEs, autofluorescence and renal function

Esther G. Gerrits1, Andries J. Smit2 and Henk J. G. Bilo1,2

1 Diabetes Center, Isala Clinics, Zwolle 2 Department of Medicine, University Medical Center Groningen, University of Groningen, the Netherlands

Correspondence and offprint requests to: Esther G. Gerrits, Isala Clinics, PO Box 10400, 8000 GK Zwolle, The Netherlands. Tel: +31384242518; E-mail: e.g.gerrits@isala.nl

Keywords: fluorescence; glycation end products; renal function



   Introduction
Top
 Introduction
Conclusion
 References
 
Accelerated formation and accumulation of AGEs occur under circumstances of hyperglycaemic or oxidative stress in age-related and chronic diseases like diabetes mellitus, chronic renal failure, neurodegenerative diseases, osteoarthritis and non-diabetic atherosclerosis [1–5]. Accumulation of irreversibly formed and chemically stable AGEs occurs on long-lived proteins such as collagen in the skin, but also in vascular basement membranes. This affects their structure and function resulting in vascular damage. Adequate renal clearance capacity is an important factor in the effective removal of AGEs. In renal failure, there is a profound decrease in clearance of AGE free adducts, which are formed mainly from proteolysis of glycated proteins. Plasma levels of these products are up to 40-fold higher in haemodialysis patients compared to healthy subjects. Increased levels of AGE-free adducts in plasma is also a characteristic of acute and chronic renal failure, whereas accumulation of AGE residues on plasma proteins appears to be limited to chronic renal failure [6–8]. Generally, AGE residues on plasma proteins are not decreased during a dialysis session, while AGE-free adducts are indeed removed by haemodiafiltration or other dialysis procedures [7]. Little is known about tissue accumulation of AGEs on long-lived proteins in patients with chronic renal failure and patients on haemodialysis.

Furthermore, both uraemic toxicity and some modalities of renal replacement therapy contribute to increased oxidative stress, inducing protein modification, which either directly or indirectly contribute to the increased formation of AGEs [2,9]. Since tissue accumulation of AGEs on long-lived proteins is a long-term process, quantitation of the collagen-bound AGEs could reflect ‘metabolic memory’ over several years. Up till recently, skin biopsies were needed to properly assess the level of tissue AGE accumulation; as this is an invasive and time-intensive method, it is not feasible in daily practice.

The autofluorescence reader
The application of a newly developed noninvasive device, the autofluorescence reader (AFR), gives the opportunity to measure skin autofluorescence (AF), which has been shown to be reasonably well correlated with skin content of some AGE residues. The technique has been validated against AGE measurements in skin biopsies from the site of the skin AF measurement, taken in patients on haemodialysis, patients with diabetes mellitus and healthy controls. Skin AF correlates with skin levels of some AGE residues: N{varepsilon}-carboxymethyl-lysine (CML), r = 0.55 (P < 0.001); pentosidine, r = 0.55 (P < 0.001); N{varepsilon}-carboxyethyl-lysine (CEL), r = 0.47 (P = 0.002) [10,11]. Thus, compared to this laborious and time intensive invasive technique and to serum or plasma AGE measurements, the AFR allows for a noninvasive and complete automated measurement within 30 s with immediate presentation of the result, which makes this device suitable for clinical application. The AFR illuminates the volar side of a skin surface of the arm of ~4 cm2, guarded against surrounding light (Figure 1a).


Figure 1
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  		Fig. 1 Autofluorescence reader in a clinical setting (a). Schematic view of the autofluorescence reader (b).

 
The principle of skin AF is based on the fluorescent properties of certain AGEs. The excitation light source has a peak intensity of ~370 nm, and emission light and reflected light from the skin are measured with a spectrometer in the range 300–600 nm (Figure 1b). Skin AF is calculated by dividing the average light intensity of the emission spectrum by the average light intensity of the excitation spectrum and is expressed in arbitrary units. Skin reflection is taken into account by using an internal reflection standard. Managing the instrument does not require special training or skills, and needs no special preparation of the subjects. Reproducibility of the device has been tested in different study populations and showed a mean relative error in skin AF of ~5%. Mean age-corrected skin AF per measuring month, per examiner and per AFR system did not differ significantly as well [12]. An important limitation of the original AFR was the inability to measure people with a dark skin type because of the high absorption grade of the excitated light. Recent developments of the AFR device have reduced this limitation to a more limited range of people with a very dark skin type. There are other limitations of skin AF measurement. Non-fluorescent AGEs are not detected—by definition—with skin AF, while the fluorescence of other non-AGE tissue components in the same range of wavelength may act like confounders. Not all AGEs show fluorescent characteristics: hydroimidazolones, CML and CEL are important AGEs, but not fluorescent. Furthermore, as already mentioned, the fluorescence patterns used by the AFR are not specific for fluorescent AGEs only, but could also be due to other fluorophores like nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). There could also be a contribution of the fluorescent oxidation adduct N-formyl-kynurenine [13,14].

In validation studies, skin AF correlated with the specific AGE skin levels of pentosidine, CML and CEL. Both fluorescent and non-fluorescent skin AGE levels correlated with each other, as indicated by the correlation between pentosidine and CML (r = 0.46; P < 0.001) [10,11]. Despite the reasonable correlation between skin AF and skin biopsy AGE content, results still have to be interpreted with the mentioned limitations and pitfalls in mind, and perhaps more detailed validation of this technique is required.

Clinical evidence
Skin AF has already been shown to be related to age, smoking and diabetes mellitus. In type 2 diabetes patients, skin AF is related to HbA1c, diabetes duration, microalbuminuria and diabetic complications [12,15]. Skin AF has also shown its predictive value for mortality and the development of microvascular disease in type 2 diabetes mellitus, as well as for mortality in haemodialysis patients [16–19]. Cross-sectional and longitudinal studies, measuring skin AF, are ongoing in other patient groups, in order to find out whether skin AF is a predictor for the development of morbidity or mortality in those patient groups as well.

AGEs and renal disease
Besides hyperglycaemia and increased oxidative stress, decreases in glomerular filtration rate (GFR) appear to be an important determinant contributing to the accumulation of AGEs. As already mentioned, a decline in renal function leads to elevated serum AGE levels. Such a relationship was confirmed in diabetes mellitus, where progressive nephropathy with decreased renal function was associated with increased accumulation of AGEs [20].

Skin AF in renal failure is not only associated with but also strongly predictive for cardiovascular disease, as shown by the independent predictive value of skin AF for total and cardiovascular mortality in haemodialysis patients [19]. As for cardiovascular dysfunction in haemodialysis patients, it has been shown that plasma AGEs were not associated with diastolic function, while skin AF was independently associated with diastolic function [21]. Prevention of the decline in renal function will probably lead to a reduced accumulation rate of AGEs, which in turn might contribute to a less dire cardiovascular outcome in patients with renal disease. The relative importance of this factor compared to other already known risk factors still awaits proper assessment, however.

Conventional methods of renal replacement therapy are only partially effective with regard to AGE clearance; the degree of removal is also dependent on the frequency and duration of dialysis [22,23]. Also, treatment itself may contribute to AGE accumulation; oxidative stress is an important factor leading to AGE formation, and some haemodialysis membranes—depending on their degree of biocompatibility—will probably contribute to increased AGE formation [24]. On the other hand, new technologies concerning certain high-flux membranes, vitamin E-coated low-flux dialyzers and convective therapies may lead to less oxidative stress and enhanced AGE removal in haemodialysis patients [25]. Preliminary evidence suggests that high-flux haemodialysis and the use of low glucose dialysates in peritoneal dialysis are associated with lower levels of skin AF (Arsov Z et al. and McIntyre N et al., unpublished data).

Renal transplantation results in a decrease in AGE accumulation, though AGE levels remain well above those of controls. Moreover, the degree of AGE accumulation could be involved in the development of cardiovascular disease and chronic renal transplant dysfunction after renal transplantation [26,27]. Increased levels of skin AF are associated with several risk factors for chronic renal transplant dysfunction and cardiovascular disease [28]. Unpublished data show an independent predictive value of skin AF for the development of chronic transplant dysfunction, which converge with the pathophysiological mechanism of oxidative stress and AGE accumulation in the outcome of graft loss in renal transplant recipients [29]. Therefore, it can be hypothesized that preventive therapy with AGE inhibitors might be helpful in preserving renal function in these transplant patients. Again it should be stressed that this hypothesis needs confirmation, and the relative importance of these risk factors weighed against the impact of other risk factors.

Skin AF and renal function in a screening setting: are skin AF levels directly related to the degree of renal failure?
Indeed, skin AF is correlated to the estimated GFR (eGFR) category, calculated with the Modification of Diet in Renal Disease (MDRD) formula (using the re-expressed four-variable MDRD) [30], when performing global tests, as shown in the example presented below (Table 1; hitherto, unpublished data). The MDRD was used as a screening instrument in a large cohort of subjects with type 2 diabetes mellitus (n = 973), participating in the ZODIAC trial.


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  		Table 1 Skin AF and MDRD

 
At first sight, such results appear to vindicate the supposed correlation between eGFR and skin AF. However, one has to keep in mind in this assessment that the MDRD formula has not been sufficiently validated as a screening tool in subjects older than 70 years old (43% of our study population). Secondly, one should bear in mind that age plays an important role in the MDRD formula, and that age in itself is one of the factors related to AGE accumulation. For statistical reasons, the eGFR cannot be corrected for age to allow a more reliable assessment. Therefore, we divided the same cohort into five groups according to age, and dichotomized the results under and above the median of eGFR (Table 2). Figure 2 shows the correlation between skin AF and MDRD for the entire patient group. As can be seen, the dichotomized results do not show a definite and consistent correlation between AFR readings and eGFR. Furthermore, Figure 2 speaks for itself when assessing the relationship between skin AF and eGFR in a large cohort. The r-square value of 0.106 (P < 0.001) is rather disappointing. These results suggest that skin AF is not a factor that is strongly associated with renal function, at least not in a screening setting in patients with type 2 diabetes.


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  		Table 2 Skin AF and eGFR in dichotomized age groups (I = mean skin AF in patients below the median of the eGFR; II = mean skin AF in patients above the median of the eGFR)

 

Figure 2
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  		Fig. 2 Scatterplot MDRD versus skin AF.

 


   Conclusion
Top
 Introduction
 Conclusion
References
 
Skin AF is a new measurement that may have prognostic utility. This noninvasive and non-time-consuming method has been studied mainly in end-stage renal disease and diabetes mellitus, but studies are ongoing in other patient groups as well. Based on the published (but still partly incomplete) evidence, skin AF, as assessed in different disease states: diabetes mellitus, renal failure, rheumatoid arthritis, is related to and predictive of morbidity and mortality.

The significance of skin AF as a meaningful tool in screening whole populations remains to be defined yet. More research is still needed in other patient populations in order to further delineate the exact role of both tissue AGEs and skin AF under various conditions. More transversal and longitudinal studies need to be done before the AFR can be used as a risk assessment tool in individual patient care. However, the potential exists. Maybe in the future, when drugs reducing AGE formation, AGE breakers or dialysis modalities reducing AGE formation will become part of the therapeutic inventory, skin AF could offer a tool to identify responders to therapy and to monitor treatment as well.

Conflict of interest statement. The first author (E. G. Gerrits) declares that she participated in the data collection, analysis, interpretation and writing of the report. She has seen and approved the final version. She has no conflicts of interest. As the corresponding author, she had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis and he had final responsibility for the decision to submit for publication. The second author (A. J. Smit) declares that he participated in the design, analysis and writing of the study that resulted in the article: Advanced Glycation Endproducts, Autofluorescence and Renal Function, by E. G. Gerrits, A. J. Smit and H. J. G. Bilo. He has seen and approved the final version. He may have a possible conflict of interest since he has been co-inventor in a patent application concerning the autofluorescence reader, and he is a stock holder in the company Diagnoptics Technologies BV (the Netherlands), co-founded by him in 2003, which develops and produces the AGE reader that is based on the prototype discussed in the present article. The third author (H. J. G. Bilo) declares that he participated in the study design, data collection, analysis, interpretation and writing of the article. He has seen and approved the final version. He has no conflicts of interest.



   References
Top
 Introduction
 Conclusion
 References
 

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Received for publication: 30. 1.08
Accepted in revised form: 21.10.08


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