Proteomic analysis of kidneys from selenoprotein M transgenic rats in response to increased bioability of selenium

Abstract

Background: To characterize changes in global protein expression in kidneys of transgenic rats overexpressing human selenoprotein M (SelM) in response to increased bioabivility of selenium (Sel), total proteins extracted from kidneys of 10-week-old CMV/hSelM Tg and wild-type rats were separated by 2-dimensional gel electrophoresis and measured for changes in expression.

Results: Ten and three proteins showing high antioxidant enzymatic activity were up- and down-regulated, respectively, in SelM-overexpressing CMV/hSelM Tg rats compared to controls based on an arbitrary 2-fold difference. Up-regulated proteins included LAP3, BAIAP2L1, CRP2, CD73 antigen, PDGF D, KIAA143 homolog, PRPPS-AP2, ZFP313, HSP-60, and N-WASP, whereas down-regulated proteins included ALKDH3, rMCP-3, and STC-1. After Sel treatment, five of the up-regulated proteins were significantly increased in expression in wild-type rats, whereas there were no changes in CMV/hSelM Tg rats. Only two of the down-regulated proteins showed reduced expression in wild-type and Tg rats after Sel treatment.

Conclusions: These results show the primary novel biological evidences that new functional protein groups and individual proteins in kidneys of Tg rats relate to Sel biology including the response to Sel treatment and SelM expression.

Figure 1

Characterization of CMV/GFP-hSelM Tg rats. (A) Expression of SelM proteins in kidney of CMV/hSelM Tg rats using antibody for both rat and human SelM. (B) Immunostaining analysis of SelM expression at 200x magnification. (C) Concentration of total antioxidants in serum by ELISA. Activities of GPx (D) and SOD (E) were detected in kidney tissues collected from CMV/EGFP-hSelM Tg and non-Tg rats. Six rats per group were assayed by ELISA. Data represent the mean ± SD of three replicates. a, p<0.05 is the significance level compared with non-Tg rats. b, p<0.05 is the significance level compared with the vehicle-treated group.

Figure 2

2-DE protein patterns in kidney tissues from CMV/EGFP-hSelM Tg and non-Tg rats. Kidney lysates (1 mg) from four groups, including vehicle-treated non-Tg rats (A), Sel-treated non-Tg rats (B), vehicle-treated CMV/hSelM Tg rats (C), and Sel-treated CMV/hSelM Tg rats (D), were subjected to one-dimensional IEF using 24- cm IPG strips in a pH range from 3–10 (nonlinear). Two-dimensional SDS-PAGE was performed on 8–18 % linear gradient acrylamide gels in an EttanDalt system. Protein spots were visualized by staining with Coomassie blue G-250.

Figure 3

Gel enlargement image showing LAP3 and BAIAP2L1 in kidney extracts. (A) Up-regulated protein spots of LAP3 and BAIAP2L1 were detected in kidney extracts from the four experimental groups. Spots differentially expressed on 2-DE were further analyzed using a matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. (B) Expression levels of two proteins regulated by Sel treatment and SelM expression are represented relative to the non-Tg group. Data represent the mean ± SD of three replicates. a, p<0.05 is the significance level compared with non-Tg rats. b, p<0.05 is the significance level compared with the vehicle-treated group.

Figure 4

Gel enlargement image showing KIAA1134 homolog, CD73 antigen, and CRP2 in kidney extracts. (A) Up-regulated protein spots of KIAA1134 homolog, CD73 antigen, and CRP2 were detected in kidney extracts from the four experimental groups. Spots differentially expressed on 2-DE were further analyzed using a matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. (B) Expression levels of two proteins regulated by Sel treatment and SelM expression are represented relative to the non-Tg group. Data represent the mean ± SD of three replicates. a, p<0.05 is the significance level compared with non-Tg rats. b, p<0.05 is the significance level compared with the vehicle-treated group.

Figure 5

Gel enlargement image showing PDGF D and PAP41 in kidney extracts. (A) Up-regulated or maintained protein spots of PDGF D and PAP41 were detected in kidney extracts from the four experimental groups. Spots differentially expressed on 2-DE were further analyzed using a matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. (B) Expression levels of two proteins regulated by Sel treatment and SelM expression are represented relative to the non-Tg group. Data represent the mean ± SD of three replicates. a, p<0.05 is the significance level compared with non-Tg rats. b, p<0.05 is the significance level compared with the vehicle-treated group.

Figure 6

Gel enlargement image showing ZFP313, HSP-60, and N-WASP in kidney extracts. (A) Three spots (ZFP313, HSP-60, and N-WASP) up-regulated only by SelM overexpression were detected in kidney extracts from the four experimental groups. Spots differentially expressed on 2-DE were further analyzed using a matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. (B) Expression levels of two proteins regulated by Sel treatment and SelM expression are represented relative to the non-Tg group. Data represent the mean ± SD of three replicates. a, p<0.05 is the significance level compared with non-Tg rats. b, p<0.05 is the significance level compared with the vehicle-treated group.

Figure 7

Gel enlargement image showing STC-1, rMCP-5, and ALKDH3 in kidney extracts. (A) Differentially regulated protein spots of STC-1, rMCP-5, and ALKDH3 were detected in kidney extracts from the four experimental groups. Spots differentially expressed on 2-DE were further analyzed using a matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. (B) Expression levels of two proteins regulated by Sel treatment and SelM expression are represented relative to the non-Tg group. Data represent the mean ± SD of three replicates. a, p<0.05 is the significance level compared with non-Tg rats. b, p<0.05 is the significance level compared with the vehicle-treated group.

Figure 8

Verification of ALKDH3, HSP-60, and LAP3 protein expression. Nitrocellulose membranes transferring 50 μg of protein from kidneys of CMV/hSelM Tg and non-Tg rats were incubated with antibody specific for ALKDH3, HSP-60, LAP3, or actin, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG. Data represent the mean ± SD of three replicates. a, p<0.05 is the significance level compared with non-Tg rats. b, p<0.05 is the significance level compared with the vehicle-treated group.