Morphological changes in diabetic kidney are associated with increased O-GlcNAcylation of cytoskeletal proteins including α-actinin 4

Abstract Purpose The objective of the present study is to identify proteins that change in the extent of the modification with O-linked N-acetylglucosamine (O-GlcNAcylation) in the kidney from diabetic model Goto-Kakizaki (GK) rats, and to discuss the relation between O-GlcNAcylation and the pathological condition in diabetes. Methods O-GlcNAcylated proteins were identified by two-dimensional gel electrophoresis, immunoblotting and peptide mass fingerprinting. The level of O-GlcNAcylation of these proteins was examined by immunoprecipitation, immunoblotting and in situ Proximity Ligation Assay (PLA). Results O-GlcNAcylated proteins that changed significantly in the degree of O-GlcNAcylation were identified as cytoskeletal proteins (α-actin, α-tubulin, α-actinin 4, myosin) and mitochondrial proteins (ATP synthase β, pyruvate carboxylase). The extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunofluorescence and in situ PLA studies revealed that the levels of O-GlcNAcylation of actin, α-actinin 4 and myosin were significantly increased in the glomerulus and the proximal tubule of the diabetic kidney. Immunoelectron microscopy revealed that immunolabeling of α-actinin 4 is disturbed and increased in the foot process of podocytes of glomerulus and in the microvilli of proximal tubules. Conclusion These results suggest that changes in the O-GlcNAcylation of cytoskeletal proteins are closely associated with the morphological changes in the podocyte foot processes in the glomerulus and in microvilli of proximal tubules in the diabetic kidney. This is the first report to show that α-actinin 4 is O-GlcNAcylated. α-Actinin 4 will be a good marker protein to examine the relation between O-GlcNAcylation and diabetic nephropathy.</p

Proteomic analysis in usual and nonspecific interstitial pneumonia

Differentiating nonspecific interstitial pneumonia (NSIP) from usual interstitial pneumonia (UIP) is important for the determination of both treatment and prognosis. Using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), we examined 8 UIPs, 8 NSIPs, and 30 normal lung tissues. Comparisons with control in 2D-DIGE showed that (a) in UIP, nine protein spots were significantly upregulated and seven were significantly downregulated, (b) in NSIP, four protein spots were significantly upregulated and nine were significantly downregulated. The detected proteins were analyzed by MALDI-TOF mass spectrometry, allowing qualitative differences in vimentin subtypes to be characterized. One vimentin subtype was upregulated in UIP, while another one was downregulated in NSIP (vs. control). These different characteristics were partially supported by the results of Western blot analysis. Our immunohistochemistry revealed vimentin expression within fibroblasts (a) in fibroblastic foci in UIP and (b) in fibrotic alveolar walls in NSIP. Differences in vimentin subtypes may provide useful biomarkers for separating NSIP from UIP, alongside differences in histological characteristics

Proteomic analysis of the lung in rats with hypobaric hypoxia-induced pulmonary hypertension

Experimental pulmonary hypertension that develops in hypobaric hypoxia is characterized by structural remodeling of the lung. Proteomics - which may be the most powerful way to uncover unknown remodeling proteins involved in enhancing cardiovascular performance - was used to study 150 male Wistar rats housed for up to 21 days in a chamber at the equivalent of 5500 m altitude level. After 14 days’ exposure to hypobaric hypoxia, pulmonary arterial pressure (PAP) was significantly increased. In lung tissue, about 140 matching protein spots were found among 8 groups (divided according to their hypobaric period) by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (pH4.5-pH6.5, 30 kDa-100 kDa). In hypobaric rats, three spots were increased two-fold or more (vs. control rats) in two-dimensional differential in-gel electrophoresis (2D-DIGE). The increased proteins were identified, by matrix-assisted laser desorption ionization time of flight (MALDI-TOF), as one isoform of heat shock protein 70 (HSP70) and two isoforms of protein disulfide isomerase associated 3. This result was confirmed by Western blotting analysis of 2D-PAGE. Conceivably, HSP70 and PDIA3 may play roles in modulating the lung structural remodeling that occurs due to pulmonary hypertension in hypobaric hypoxia