Alterations in fatty acid utilization and an impaired antioxidant defense mechanism are early events in podocyte injury: a proteomic analysis - PubMed (original) (raw)
Alterations in fatty acid utilization and an impaired antioxidant defense mechanism are early events in podocyte injury: a proteomic analysis
Corina Mayrhofer et al. Am J Pathol. 2009 Apr.
Abstract
Ultrastructural alterations of podocytes are closely associated with loss of glomerular filtration function. In the present study, we explored changes at the proteome level that paralleled the disturbances of podocyte architecture in the early stages of puromycin aminonucleoside (PA) nephrosis in vivo. Using two-dimensional fluorescence difference gel electrophoresis and vacuum matrix-assisted laser desorption/ionization mass spectrometry combined with postsource decay fragment ion analysis and high-energy collision-induced dissociation tandem mass spectrometry, 23 differentially expressed protein spots, corresponding to 16 glomerular proteins that are involved in various cellular functions, were unambiguously identified, and a subset was corroborated by Western blot analysis. The majority of these proteins were primarily related to fatty acid metabolism and redox regulation. Key enzymes of the mitochondrial beta-oxidation pathway and antioxidant enzymes were consistently down-regulated in PA nephrosis. These changes were paralleled by increased expression levels of CD36. PA treatment of murine podocytes in culture resembled these specific protein changes in vitro. In this cell system, the modulatory effects of albumin-bound fatty acids on the expression levels of Mn-superoxide dismutase in response to PA were demonstrated as well. Taken together, these results indicate that a disrupted fatty acid metabolism in concert with an impaired antioxidant defense mechanism in podocytes may play a role in the early stages of PA-induced lesions in podocytes.
Figures
Figure 1
Proteomics study. A: Transmission electron micrographs showing a normal rat glomerular capillary (left) and a PAN glomerular capillary (right). Arrows indicate the podocyte foot processes and arrowheads mark the presence of large vacuoles in podocytes after PA administration. Proteins extracted from isolated normal control and PAN glomeruli were then labeled with Cy5 and Cy3, respectively, and further separated on one 2-D gel and detected by fluorescence scanning of the gel according to the appropriate wavelengths. B: Silver-stained 2-D pattern of extracted glomerular proteins separated on a pH range from 3 to 10 nonlinear in the first dimension. The second dimension was performed on a 3.6 to 15% self-made gradient SDS-PAA gel. The numbered protein spots were identified by vMALDI mass spectrometry. The identified proteins are listed in Table 1. C: Verification of the DIGE results by Western blot analysis using antibodies against HCDH, PRDX-3, and Mn-SOD. Total glomerular lysates from normal control and PAN animals were separated by 1-D SDS-PAGE and then transferred onto a nitrocellulose membrane. Representative Western blots are given in the left panels and the data presented in the right panels are means ± SD. The expression of GAPDH was used for normalization. The ratios in the control group were considered as 1. *P ≤ 0.05 with respect to control. Abbreviations: cl, capillary lumen; us, urinary space.
Figure 2
Detection of infiltrating macrophages/monocytes in glomeruli. A: Phase contrast micrographs and the corresponding fluorescence micrographs of a representative normal rat kidney section and a representative kidney section after PA treatment. Two-μm sections were stained for macrosialin (CD68), a macrophage-specific protein. Nuclei were stained with DAPI. Arrowheadsmark the localization of CD68-positive cells. B: Number of intraglomerular CD68-positive cells. The data presented are means ± SD. Original magnifications, ×20.
Figure 3
Effect of PA on CD36. Western blot analysis of CD36 expression using glomerular lysates from control normal as well as PA-treated animals. A representative Western blot is given in A and the data presented in B are means (n = 3 in each group) ± SD. The expression of GAPDH was used for normalization. The ratios in the control group were considered as 1. *P ≤ 0.05 with respect to control.
Figure 4
Effect of PA on proteins involved in fatty acid metabolism in podocytes in vitro. Cultivated podocytes were either untreated or treated with 5 μg/ml of PA for 24 or 48 hours and the protein expression of HCDH (A), CD36 (B), and UCP3 (C) was examined by Western blotting. In each case a representative Western blot is given in the top panel and the data presented in the bottom panel are means ± SD. The expression of GAPDH was used for normalization. The ratios in the control group were considered as 1. *P ≤ 0.05 and **P ≤ 0.01 with respect to control.
Figure 5
Subcellular localization of CD36. A: Fluorescence micrograph of cultured podocytes that were exposed to PA for 24 hours. Cells were fixed, permeabilized, and stained with CD36 (diluted 1:200 and incubated for 1 hour at room temperature) using an Alexa Fluor 488-conjugated secondary antibody (diluted 1:1000 and incubated for 1 hour at room temperature). Nuclei were stained with DAPI. To verify the specificity of the antibody, cells were incubated with unspecific rabbit IgG. B: Podocytes that were exposed to PA for 24 hours were biotin-labeled on the cell surface and protein extracts (TL) without and after purification (P) with NeutrAvidin gel were separated by 1-D SDS-PAGE and transferred onto nitrocellulose. Western blotting was performed using antibodies against CD36, Mn-SOD, and α-DG. The positions of molecular mass markers are indicated at the right. Original magnifications, ×40.
Figure 6
PA induced-changes in the protein expression of Mn-SOD. A: Podocytes were either untreated or treated with PA for 24 and 48 hours in regular culture medium containing 10% FCS and the protein expression of Mn-SOD was examined by Western blotting. B: Podocytes cultured in medium containing 0.1% FCS either alone or supplemented with 0.1% fatty acid (FA)-free BSA or 0.1% FA bound to albumin exposed to PA for 48 hours. The values are the means ± SD. The expression of GAPDH was used for normalization. The ratio in the control group was considered as 1. *P ≤ 0.05 and **P ≤ 0.01 with respect to control.
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