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

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 ironmediated toxicity

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BACKGROUND AND AIMS:The proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn2+ and Cu2+ concentration during hemodialysis. METHODS AND RESULTS:Epitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn2+ or Cu2+ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p<0.001). During hemodialysis the relative proportion of RYSK173 CN-1 decreased in parallel with increased serum Zn2+ and Cu2+ concentrations after dialysis. CONCLUSIONS:Our study clearly indicates that RYSK173 recognizes a sequence within the transition metal binding site of CN-1, thus supporting our hypothesis that metal binding to CN-1 masks the epitope. The CN-1 RYSK173 proportion appears overall increased in ESRD patients, yet it decreases during hemodialysis possibly as a consequence of a relative increase in transition metal bound enzyme

Recovery of recombinant CN-1 in serum.

<p>A: Equal amounts of recombinant CN-1 were spiked in PBS, human serum or FCS. While in human serum detection of CN-1 by RYSK173 was strongly diminished this was not observed in FCS. B: The influence of ZnCl₂ (100μM) and CuSO₄ (100μM) on detection of CN-1 by RYSK173 was tested. Ns: not significant.</p

Serum CN-1 concentrations in ESRD patients.

<p>A: Sera of 31 ESRD patients were collected and tested in the ATLAS based and RYSK173 based ELISA. Sera of 111 healthy individuals served as control. The results are expressed as RYSK173/ATLAS ratio x 100%. B: Serum was collected directly before or after hemodialysis. The influence of hemodialysis on total CN-1 expression (B), the RYSK173 proportion (C) and changes in Zn²⁺ and Cu²⁺ (D) concentrations were assessed. UF: ultrafiltration, BW: body weight, HD: hemodialysis, conc: concentration.</p

Epitope mapping of RYSK173.

<p>A: Myc-tagged recombinant CN-1 fragments were expressed in COS7 cells by transfection. Cells were harvested and the recombinant proteins were detected by Westernblotting using RYSK173 or anti-Myc (Fig on the left). Three overlapping synthetic peptides (CN1-1, CN1-2 and CN1-3) corresponding to bp 313–471 were tested in dotblot analysis (Fig on the right). B: Two additional peptides (CN1-4 and CN1-5) corresponding to the overlapping sequence of CN1-2 and CN1-3 were tested in ELISA for recognition by RYSK173. C: Location of the dinuclear zinc center at the CN-1 homodimer interface. The monomer on the left is depicted as a ribbon drawing and the monomer on the right is rendered as solid surface. Zinc1 and zinc2 in the left monomer are shown as cyan and magenta colored spheres, respectively. The enlarged view details the position of the RYSK173 epitope, which is confined to the amino acid sequence H132 to D139 (both marked with an asterisk). The surface of the left subunit is rendered semi-transparent. The Fig was made with Protein Workshop. D: H132 was exchanged for glutamine (Q) by site directed mutagenesis. The recombinant proteins were detected in transfected cell lysates using the ATLAS and RYSK173 based ELISA. The results are expressed as RYSK173/ATLAS ratio x 100%. NC: negative control, WT: wild type.</p

Recognition of recombinant and serum CN-1 by RYSK173.

<p>A: recombinant CN-1 was expressed in HUVECs by lentiviral transduction. The recombinant proteins were detected in cell lysates and supernatants using the ATLAS (filled bars) and RYSK173 (open bars) based ELISA. The results of 6 transduction experiments are depicted and expressed as mean CN-1 concentration (ng/ml) ± SD. B: Serum samples of 111 healthy individuals were tested in the ATLAS and RYSK173 based ELISA. C: Cell disruption was performed by repeated freeze thawing. Similar as in serum RYSK173 detected significantly lower amounts of CN-1.</p