Tim Weigand1, Benjamin Singler1, Thomas Fleming2, Peter Nawroth2,3,4, Karel D Klika5, Christian Thiel1, Hans Baelde6, Sven F Garbade1, Andreas H Wagner7, Markus Hecker7, Benito A Yard8, Albert Amberger9, Johannes Zschocke9, Claus P Schmitt1, Verena Peters1. 1. Centre for Paediatric and Adolescent Medicine, University Hospital of Heidelberg, Heidelberg, Germany. 2. Department of Medicine I and Clinical Chemistry, University Hospital of Heidelberg, Heidelberg, Germany. 3. German Center for Diabetes Research (DZD), Neuherberg, Germany. 4. Institute for Diabetes and Cancer IDC Helmholtz Center Munich, Germany & Joint Heidelberg-IDC Translational Diabetes Program, Neuherberg, Germany. 5. Core Facility, Molecular Structure Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany. 6. Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands. 7. Department of Cardiovascular Physiology, Heidelberg University, Heidelberg, Germany. 8. Department of Nephrology, Endocrinology and Rheumatology, Fifth Department of Medicine, Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Germany. 9. Division of Human Genetics, Medical University Innsbruck, Innsbruck, Austria.
Abstract
BACKGROUND/AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MG), contribute to diabetic complications. MG-scavenging capacities of carnosine and anserine, which have been shown to mitigate diabetic nephropathy, were evaluated in vitro and in vivo. METHODS: MG-induced cell toxicity was characterized by MTT and MG-H1-formation, scavenging abilities by Western Blot and NMR spectroscopies, cellular carnosine transport by qPCR and microplate luminescence and carnosine concentration by HPLC. RESULTS: In vitro, carnosine and anserine dose-dependently reduced N-carboxyethyl lysine (CEL) and advanced glycation end products (AGEs) formation. NMR studies revealed the formation of oligo/polymeric products of MG catalyzed by carnosine or anserine. MG toxicity (0.3-1 mM) was dose-dependent for podocytes, tubular and mesangial cells whereas low MG levels (0.2 mM) resulted in increased cell viability in podocytes (143±13%, p<0.001) and tubular cells (129±3%, p<0.001). Incubation with carnosine/anserine did not reduce MG-induced toxicity, independent of incubation times and across large ranges of MG to carnosine/anserine ratios. Cellular carnosine uptake was low (<0.1% in 20 hours) and cellular carnosine concentrations remained unaffected. The putative carnosine transporter PHT1 along with the taurine transporter (TauT) was expressed in all cell types while PEPT1, PEPT2 and PHT2, also belonging to the proton-coupled oligopeptide transporter (POT) family, were only expressed in tubular cells. CONCLUSION: While carnosine and anserine catalyze the formation of MG oligo/polymers, the molar ratios required for protection from MG-induced cellular toxicity are not achievable in renal cells. The effect of carnosine in vivo, to mitigate diabetic nephropathy may therefore be independent upon its ability to scavenge MG and/or carnosine is mainly acting extracellularly.
BACKGROUND/AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MG), contribute to diabetic complications. MG-scavenging capacities of carnosine and anserine, which have been shown to mitigate diabetic nephropathy, were evaluated in vitro and in vivo. METHODS:MG-induced cell toxicity was characterized by MTT and MG-H1-formation, scavenging abilities by Western Blot and NMR spectroscopies, cellular carnosine transport by qPCR and microplate luminescence and carnosine concentration by HPLC. RESULTS: In vitro, carnosine and anserine dose-dependently reduced N-carboxyethyl lysine (CEL) and advanced glycation end products (AGEs) formation. NMR studies revealed the formation of oligo/polymeric products of MG catalyzed by carnosine or anserine. MG toxicity (0.3-1 mM) was dose-dependent for podocytes, tubular and mesangial cells whereas low MG levels (0.2 mM) resulted in increased cell viability in podocytes (143±13%, p<0.001) and tubular cells (129±3%, p<0.001). Incubation with carnosine/anserine did not reduce MG-induced toxicity, independent of incubation times and across large ranges of MG to carnosine/anserine ratios. Cellular carnosine uptake was low (<0.1% in 20 hours) and cellular carnosine concentrations remained unaffected. The putative carnosine transporter PHT1 along with the taurine transporter (TauT) was expressed in all cell types while PEPT1, PEPT2 and PHT2, also belonging to the proton-coupled oligopeptide transporter (POT) family, were only expressed in tubular cells. CONCLUSION: While carnosine and anserine catalyze the formation of MG oligo/polymers, the molar ratios required for protection from MG-induced cellular toxicity are not achievable in renal cells. The effect of carnosine in vivo, to mitigate diabetic nephropathy may therefore be independent upon its ability to scavenge MG and/or carnosine is mainly acting extracellularly.
Authors: Tim Weigand; Florian Colbatzky; Tilman Pfeffer; Sven F Garbade; Kristina Klingbeil; Florian Colbatzky; Michael Becker; Johanna Zemva; Ruben Bulkescher; Robin Schürfeld; Christian Thiel; Nadine Volk; David Reuss; Georg F Hoffmann; Marc Freichel; Markus Hecker; Tanja Poth; Thomas Fleming; Gernot Poschet; Claus P Schmitt; Verena Peters Journal: Int J Mol Sci Date: 2020-07-10 Impact factor: 5.923
Authors: Xinmiao Zhang; Angelica Rodriguez-Niño; Diego O Pastene; Prama Pallavi; Jacob van den Born; Stephan J L Bakker; Bernhard K Krämer; Benito A Yard Journal: Sci Rep Date: 2021-04-13 Impact factor: 4.379
Authors: Rekha Sharma; Renuka Sehrawat; Sonika Ahlawat; Vivek Sharma; Alka Parmar; M S Thakur; A K Mishra; M S Tantia Journal: Sci Rep Date: 2022-03-03 Impact factor: 4.379