Hyperglycemia Does Not Affect Iron Mediated Toxicity of Cultured Endothelial and Renal Tubular Epithelial Cells: Influence of L-Carnosine

Abstract

Iron has been suggested to affect the clinical course of type 2 diabetes (T2DM) as accompanying increased intracellular iron accumulation may provide an alternative source for reactive oxygen species (ROS). Although carnosine has proven its therapeutic efficacy in rodent models of T2DM, little is known about its efficacy to protect cells from iron toxicity. We sought to assess if high glucose (HG) exposure makes cultured human umbilical vein endothelial cells (HUVECs) and renal proximal tubular epithelial cells (PTECs) more susceptible to metal induced toxicity and if this is ameliorated by L-carnosine. HUVECs and PTECs, cultured under normal glucose (5 mM, NG) or HG (30 mM), were challenged for 24 h with FeCl3. Cell viability was not impaired under HG conditions nor did HG increase susceptibility to FeCl3. HG did not change the expression of divalent metal transporter 1 (DMT1), ferroportin (IREG), and transferrin receptor protein 1 (TFRC). Irrespective of glucose concentrations L-carnosine prevented toxicity in a dose-dependent manner, only if it was present during the FeCl3 challenge. Hence our study indicates that iron induced cytotoxicity is not enhanced under HG conditions. L-Carnosine displayed a strong protective effect, most likely by chelation of iron mediated toxicity.

Figure 1

Iron and zinc mediated toxicity in HUVECs (a) and PTECs (b). PTECs were more tolerant to iron toxicity as compared to HUVECs (c). Iron mediated toxicity was not largely influenced by transferrin (TRF) (d). Although PTECs were less susceptible to iron mediated toxicity, toxicity increased upon longer exposure (e). Iron toxicity was associated with an increased number of TUNEL positive cells, which was significantly abrogated by L-carnosine (f).

Figure 2

Influence of high glucose and carnosine on iron mediated toxicity in HUVECs (a, c, and e) and PTECs (b, d, and f). High glucose concentration neither increased cell susceptibility to iron nor disrupted the protective effect of carnosine on iron toxicity in HUVECs (a) and PTECs (b). The protective effect of carnosine showed a clear dose-dependent relation in both HUVECs (c) and PTECs (d). Carnosine was only protective when presented during iron challenge but not when cells were pretreated (e and f). Like carnosine also the iron chelator DFO prevented iron mediated toxicity in HUVEC when iron concentration was less than 600 μM (g). Med: medium, CN: carnosine, Glc: high glucose, and Glc + CN: high glucose + carnosine.

Figure 3

Ectopic expression of the human serum carnosinase-1 in HUVECs did not abrogate the protective effect of carnosine (a). The ability of the constitutive amino acids of carnosine, that is, β-alanine and L-histidine, to protect HUVECs against iron mediated toxicity was tested (b). Although L-histidine was slightly protective when iron concentration was 150 μM, protection was clearly less compared to L-carnosine. No protection was afforded by β-alanine. Recombinant carnosinase activity was attenuated by the presence of 300 μM FeCl₃ (c). CN: carnosine.

Figure 4

Influence of glucose and L-carnosine on DMT1 (a) and TFRC (b) mRNA expression. HUVECs (open bars) and PTECs (filled bars) were cultured for 24 h in the presence of 20 mM L-carnosine (CN), 60 μM of FeCl₃, or both combinations. In addition cells were cultured for 3 days in HG medium (Glc). Total RNA was isolated and DMT1 (a) and TFRC (b) mRNA expression were assessed by qPCR. In (c) protein expression of DMT1 in HUVECs and PTECs is shown.