NDT Advance Access originally published online on June 27, 2007
Nephrology Dialysis Transplantation 2007 22(10):3093-3094; doi:10.1093/ndt/gfm398
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Preparation of advanced glycation end products in vitro
Email: F.Waanders{at}int.umcg.nlSir,
With great interest we read the article by Chang et al. [1] on the enhancement of epithelial sodium channel mRNA expression and protein level in renal cortical collecting duct (CCD) cells by advanced glycation end products (AGEs). The authors show that AGEs can increase sodium uptake in CCD cells in a dose-dependent way, providing a novel mechanism for the dysregulation of sodium balance in diabetic nephropathy.
However, we have concerns on their in vitro preparation of AGEs. Since their results are dependent on the stimulation of renal CCD cells with AGEs prepared in vitro, it is very important that they prepared their AGEs properly. For instance, Valencia et al. [2] showed that AGE preparations which were free of significant levels of endotoxin contamination, failed to induce proinflammatory cellular responses, whereas endotoxin induced cell surface VCAM-1 on HMEC-4 endothelial cells and tumour necrosis factor-
secretion by primary human PBMCs. Therefore, to exclude the possibility that the effects of AGEs on epithelial sodium channel expression in renal CCD are mediated by endotoxin contamination, the authors should have used endotoxin-free bovine serum albumin (BSA) and assayed the control and AGE preparation for endotoxin contamination, by using, for example, the Limulus amebocyte lysate assay.
The authors added azide to prevent contamination by live bacteria under these pro-biotic conditions (a high glucose concentration at 37°C). They do not describe the use of sterilization filters before the 6 week incubation period nor after dialysis, which is a procedure with a risk of contamination. In our opinion, using sterilization filters is important in the process of AGE-preparation. But as mentioned earlier, the main problem is contamination by endotoxin.
In addition to the aforementioned concerns, the degree of glycation was only indirectly assessed as fluorescence measured by spectrophotometry. Although this indeed strongly suggests that AGEs have been formed, it would have been more elegant to show specific AGE levels such as N
-(carboxymethyl)lysine (CML) and N-(carboxyethyl)lysine (CEL).
The authors used the correct control preparation which was treated identically, with the exception that glucose was omitted. In our lab, we recently prepared AGEs by incubating endotoxin-free BSA 300 mg/ml at 37°C for 6 weeks with D-glucose (90 g/l) in a 0.4 M phosphate buffer at pH 7.6. Interestingly, we saw an increase in CML and CEL in our control preparation, which was treated identically but without the addition of glucose, when compared with non-incubated BSA (Figure 1). It would have been interesting to use non-incubated endotoxin-free BSA as a control as well.
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We conclude that Chang et al. propose a novel mechanism that could be involved in disturbances of sodium balance in diabetic nephropathy, i.e. an AGE-induced increase in expression of the epithelial sodium channel mRNA and protein, with enhanced sodium uptake in renal CCD cells. To substantiate this interesting hypothesis, it would be important to exclude endotoxin mediated effects.
Conflict of interest statement. None declared.
1Department of Nephrology
University Medical Centre Groningen
University of Groningen
2Department of Internal Medicine
University Hospital Maastricht
Maastricht
3Department of Pathology and Laboratory Medicine
University Medical Centre Groningen
University of Groningen, the Netherlands
Notes
Dr Chang et al. have been invited to reply to this letter, but we did not receive an answer.
References
- Chang CT, Wu MS, Tian YC, et al. Enhancement of epithelial sodium channel expression in renal cortical collecting ducts cells by advanced glycation end products. Nephrol Dial Transplant (2007) 22:722–731.
[Abstract/Free Full Text] - Valencia JV, Mone M, Koehne C, Rediske J, Hughes TE. Binding of receptor for advanced glycation end products (RAGE) ligands is not sufficient to induce inflammatory signals: lack of activity of endotoxin-free albumin-derived advanced glycation end products. Diabetologia (2004) 47:844–852.[CrossRef][Web of Science][Medline]
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