Title

 

Authors

D. Gallo
Callerio Foundation Onlus, Institutes of Biological Researches, Trieste, Italy; Dept. of Pharmacology, University of Padua, Italy

 

Abstract

Lysozyme (LZ) is an enzyme known for its antimicrobial and immunomodulating activities (Jolles, 1976). LZ has also recently shown to possess Advanced Glycation Endproducts (AGE)-sequestring properties (Zheng et al., 2001). LZ binds AGE, specifically, noncovalenly, and with high affinity (Kd=50 nM) (Li et al., 1995). AGE are a chemically heterogeneous group of macromolecules that are physiologically and non-enzymatically formed by the interactions of reducing sugars with free amino groups of proteins, lipids and nucleic acids, and their formation increases under high glucose concentration. A growing body of evidence links the sustained formation and accumulation of AGE to the pathogenesis of diabetic complications, such as vascular complications or diabetic nephropathy. A more recent work (Cocchietto et al., 2008) has demonstrated that orally administered LZ can significantly prevent a number of typical modifications of the early stage of diabetic nephropathy, i. e. microalbuminuria and glomerular hypertrophy. Among the most evident effects of AGE there is an increased formation of intracellular reactive oxygen species (ROS) (Alikhani et al. 2006). AGE can directly lead to enhanced formation of free radicals, but their effects can also result from the interactions between AGE and their receptors, among which RAGE (Receptor for Advanced Glycation Endproducts) is the most studied. On the basis of these background, the rationale of the research is that LZ could play an intracellular role in glomerular and tubular cells in the kidney, regulating a number of pathways involved in the control of oxidative stress. Due to the low amount of knowledge in these fields, the first goal of the present work is the optimization of an in vitro cellular system in which to simulate AGE-induced oxidative stress.Initially, we tested the viability and the production of intracellular ROS in two cell lines: porcine kidney cells (LLC-PK1) and adult dermal microvascular endothelial cells (ADMEC). The viability of both cell lines, tested with MTT and SRB tests, was not significantly altered after exposure to AGE. Intracellular ROS production in both cell lines seems to be not affected by AGE. These results suggest that the cell lines chosen don’t fit for the purpose. We then shifted to an alternative cellular model, represented by a human kidney cell line (HK-2) on which we analysed the expression of RAGE using RT-PCR: cells were treated with 10 µM of AGE-BSA and with an equivalent dose of Control-BSA. HK-2 cells showed the ability to produce the mRNA for RAGE. The expression of mRNA is not altered by AGE treatments. Then, we tested the production of ROS after exposure to AGE: HK-2 cells ROS production seems to be not affected by the treatments with a range of concentrations of AGE (10-30 µM) for 24 hours. This aspect is still unclear and will be the object of further studies.
It is known that AGE increase ROS production and that ROS have also a cell signalling role. In addition, more recent findings are showing the relevance of another pathway involved in cell protection against oxidative stress, the pathway of Nrf2 (Hur et al., 2011).
Then, it is reasonable to presume that LZ can play a role on signalling cascades such as MAPK, NF-kB or Nrf2, some of the most important mediators of ROS pathway.
 
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