Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells

Abstract

The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG) growth conditions as compared to isoosmotic/normal glucose control (NG(⁎)) conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1) proteins involved in cell survival/cell signaling and (2) proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB), supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase), suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.

Figure 1

Murine GMC proteome altered by acute (2 h) exposure to HG culture conditions. GMC cells were grown to 80% confluence and were serum-starved (0.5% FBS) overnight, and were treated for 2 h with 25 mM glucose (HG) or 5 mM glucose + 20 mM mannitol (NG^∗) as an isoosmotic control. Cells were lysed using 2DE buffer and 75 μg protein used for 2DE analysis. Proteins whose expression is altered by 2 h HG are annotated on the gel with identifications provided in Table 1. Data are representative of five individual gels for HG and for NG^∗ conditions.

Figure 2

Network analysis of protein expression patterns using Ingenuity Pathways Analysis. (a) The top scoring network (Network 1) addressed cancer, reproductive system disease, and hematological disease and included 25 identified proteins out of 35 total network components. The score 49 suggests the odds of 1 out of 10⁴⁹ for assembling randomly these protein identifications out of the existing murine protein database. (b) Network 2, defined by IPA, includes PHB. For (a) and (b), red indicates protein spots whose spot volume increased with 2 h high glucose. Green indicates proteins spots whose spot volume decreased with 2 h high glucose.

Figure 3

Validation of 2DE results for the enhanced PHB expression. Murine GMCs were cultured and treated for 2 h with HG and NG^∗ as described. Cells were lysed in 2DE buffer, diluted into Laemmli buffer, and used for immunoblot experiments (a) and quantification of 1DE IB experiments for PHB expression normalized to total actin expression (b). Data is presented as a mean of three experiments. Statistical analysis of differences between the means of HG and NG^∗ was achieved by t-test.

Figure 4

2DE immunoblot experiments were used to determine the effects of HG and NG^∗ on PHB isoforms. Following the transfer and development of PHB IB, images were aligned and densitometric measurements were estimated using ImageJ for the acidic one-third of the PHB charge train and for the basic two-thirds of the PHB charge train (IB images on right). Fractional values for PHB charge train components (acid and basic ends) were used to determine statistical significance differences (left bar graphs). M is the same as NG^∗ (5 mM D-glucose + mannitol); HG is 25 mM D-glucose. ^∗ p value < 0.01.

Figure 5

(a) Murine GMCs were seeded into 8-well chambered cover glass, grown, and treated as described in the methods. PHB detection was with the same primary antibody as used for IB. PHB detection with an Alexa Fluor 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Confocal software was used to estimate pixel density in GMC and in nuclei (as defined by DAPI). Nuclei pixel density was subtracted from total density and plotted (b). Differences were estimated by t-test with significance at p value < 0.05.

Figure 6

Correlative validation of 2DE results for the regulation of pathways involved in proteostasis. Immunoblot analysis of PSMA2 and PDI from mesangial cells cultured for 2 h in HG or NG^∗ medium. Expression of PSMA2 increased whereas PDI decreased following 2 h HG. Bar graphs, densitometric quantitation of PSMA2 or PDI normalized to GAPDH for each lane. Data are average ± SEM ^# p < 0.05 versus NG^∗.