Carnosine Prevents Apoptosis of Glomerular Cells and Podocyte Loss in STZ Diabetic Rats

Background/Aims: We identified carnosinase-1 (CN-1) as risk-factor for diabetic nephropathy (DN). Carnosine, the substrate for CN-1, supposedly is a protective factor regarding diabetic complications. In this study, we hypothesized that carnosine administration to diabetic rats might protect the kidneys from glomerular apoptosis and podocyte loss. Methods: We examined the effect of oral L-carnosine administration (1g/kg BW per day) on apoptosis, podocyte loss, oxidative stress, AGEs and hexosamine pathway in kidneys of streptozotocin-induced diabetic Wistar rats after 3 months of diabetes and treatment. Results: Hyperglycemia significantly reduced endogenous kidney carnosine levels. In parallel, podocyte numbers significantly decreased (-21% compared to non-diabetics,

N-Glycosylation of Carnosinase Influences Protein Secretion and Enzyme Activity: Implications for Hyperglycemia

OBJECTIVE-The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1 secretion is changed under hyperglycemic conditions. RESULTS-N-glycosylation of CN-1 was either inhibited by tunicamycin in pCSII-CN-1-transfected Cos-7 cells or by stepwise deletion of its three putative N-glycosylation sites. CN-1 protein expression, N-glycosylation, and enzyme activity were assessed in cell extracts and supernatants. The influence of hyperglycemia on CN-1 enzyme activity in human serum was tested in homozygous (CTG)(5) diabetic patients and healthy control subjects Tunicamycin completely inhibited CN-1 secretion Deletion of all N-glycosylation sites was required to reduce CN-1 secretion efficiency. Enzyme activity was already diminished when two sites were deleted. In pCSII-CN-1-transfected Cos-7 cells cultured in medium containing 25 mmol/l D-glucose, the immature 61 kilodaltons (kDa) CN-1 immune reactive band was not detected. This was paralleled by an increased GlcNAc expression in cell lysates and CN-1 expression in the supernatants. Homozygous (CTG)(5) diabetic patients had significantly higher serum CN-1 activity compared with genotype-matched, healthy control subjects CONCLUSIONS-We conclude that apart from the (CTG)(n) polymorphism in the signal peptide of CN-1, N-glycosylation is essential for appropriate secretion and enzyme activity. Since hyperglycemia enhances CN-1 secretion and enzyme activity, our data suggest that poor blood glucose control in diabetic patients might result in an increased CN-1 secretion even in the presence of the (CTG)(5) allele Diabetes 59:1984-1990, 201

Atorvastatin reduces the expression of aldo-keto reductases in HUVEC and PTEC. A new approach to influence the polyol pathway

Purpose: Increased flux of glucose via the polyol pathway, oxidative stress and ischaemia lead to the upregulation of the aldose reductase (AR), the key enzyme of the polyol pathway. This adversely affects the organism and can in part be reduced by inhibition of the enzyme. Methods: In this study, we examined the effect of the HMG-CoA-reductase inhibitor atorvastatin on the expression of aldose reductase (AR, AKR1B1), aldehyde reductase (AldR, AKR1A1) and small intestine reductase (SIR, AKR1B10) in human umbilical vein endothelial cells (HUVEC) and human proximal tubular epithelial cells (PTEC) by RT-PCR. Results: In HUVEC, atorvastatin reduces the expression of aldehyde reductase and aldose reductase compared with control medium (-20% and -12% respectively, P < 0.05), while small intestine reductase is not expressed. In PTEC no regulation of aldehyde reductase and aldose reductase by atorvastatin could be measured, while the expression of small intestine reductase was reduced by 37% compared with control medium (P < 0.05). The reduction observed was not abolished by the addition of mevalonic acid. Conclusion: The reduction of members of the aldo-keto-reductase family by atorvastatin is a novel way to influence the polyol pathway and a new pleiotropic effect of atorvastatin

Different conformational forms of serum carnosinase detected by a newly developed sandwich ELISA for the measurements of carnosinase concentrations

Serum carnosinase (CN-1) measurements are at present mainly performed by assessing enzyme activity. This method is time-consuming, not well suited for large series of samples and can be discordant to measurements of CN-1 protein concentrations. To overcome these limitations, we developed sandwich ELISA assays using different anti-CN-1 antibodies, i.e., ATLAS (polyclonal IgG) and RYSK173 (monoclonal IgG1). With the ATLAS-based assay, similar amounts of CN-1 were detected in serum and both EDTA and heparin plasma. The RYSKS173-based assay detected CN-1 in serum in all individuals at significantly lower concentrations compared to the ATLAS-based assay (range: 0.1-1.8 vs. 1-50 mu g/ml, RYSK- vs. ATLAS-based, P <0.01). CN-1 detection with the RYSK-based assay was increased in EDTA plasma, albeit at significantly lower concentrations compared to ATLAS. In heparin plasma, CN-1 was also poorly detected with the RYSK-based assay. Addition of DTT to serum increased the detection of CN-1 in the RYSK-based assay almost to the levels found in the ATLAS-based assay. Both ELISA assays were highly reproducible (R: 0.99, P <0.01 and R: 0.93, P <0.01, for the RYSK- and ATLAS-based assays, respectively). Results of the ATLAS-based assay showed a positive correlation with CN-1 activity (R: 0.62, P <0.01), while this was not the case for the RYSK-based assay. However, there was a negative correlation between CN-1 activity and the proportion of CN-1 detected in the RYSK-based assay, i.e., CN-1 detected with the RYSK-based assay/CN-1 detected with the ATLAS-based assay x 100% (Spearman-Rang correlation coefficient: -0.6, P <0.01), suggesting that the RYSK-based assay most likely detects a CN-1 conformation with low CN-1 activity. RYSK173 and ATLAS antibodies reacted similarly in Western blot, irrespective of PNGase treatment. Binding of RYSK173 in serum was not due to differential N-glycosylation as demonstrated by mutant CN-1 cDNA constructs. In conclusion, our study demonstrates a good correlation between enzyme activity and CN-1 protein concentration in ELISA and suggests the presence of different CN-1 conformations in serum. The relevance of these different conformations is still elusive and needs to be addressed in further studies