ABSTRACT
There is evidence that mediators of inflammation including components of the cytokine system are present in human and experimental diabetic kidney disease. CCAAT/enhancer-binding proteins (C/EBPs) represent a family of cytokine-inducible transcription factors. C/EBPs themselves regulate cytokine expression and also the expression of acute-phase reactants and connective tissue proteins. At least three C/EBP isoforms (alpha, beta, delta) are known. Upon stimulation with cytokines or bacterial lipopolysaccharide, the expression of the alpha isoform typically decreases, and the expression of the beta and/or delta isoforms increases. In view of the fact that components of the inflammatory response are present in diabetic kidney disease, there is a potential that the expression and activity of renal C/EBPs are altered in the diabetic state. In this study we sought to examine the status of C/EBP proteins in kidneys of rats with streptozotocin-induced diabetes mellitus. Diabetes was induced in 5 male Sprague-Dawley rats. Eight weight-matched non-diabetic rats were used as controls. Animals were sacrificed after 4 weeks, and the whole kidney nuclear protein was extracted. An electrophoretic mobility shift assay showed that DNA-binding activity was present in all five kidney nuclear extracts of the diabetic animals, but in only 2 out of 8 control samples (p < 0.05). A supershift assay showed that the DNA-bound protein complex consisted mainly of the C/EBPbeta isoform. Western analysis showed an increase of the C/EBPbeta protein in renal nuclear extracts of the diabetic animals compared to controls (p < 0.05). There was a decrease of the C/EBPalpha protein in the kidney nuclear extracts of the diabetic animals compared to controls (p < 0.05). We conclude that renal C/EBP dynamics are altered in experimental diabetes mellitus and that the patterns of C/EBP changes resemble those observed after cytokine or lipopolysaccharide stimulation.
Subject(s)
DNA-Binding Proteins/analysis , DNA-Binding Proteins/metabolism , Diabetes Mellitus, Experimental/metabolism , Kidney/chemistry , Nuclear Proteins/analysis , Nuclear Proteins/metabolism , Animals , Blood Glucose , Blotting, Western , CCAAT-Enhancer-Binding Proteins , DNA/metabolism , DNA-Binding Proteins/genetics , Kidney/metabolism , Male , Nuclear Proteins/genetics , Oligonucleotide Probes , Rats , Rats, Sprague-Dawley , Transcription Factors/analysis , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Glucosylceramide (GlcCer) and related glycosphingolipids have been implicated as causal elements in both the growth of cells and in the regulation of hormonal signaling. We therefore studied whether the renal hypertrophy induced by diabetes was associated with enhanced synthesis of glycosphingolipids. 16 d after the induction of diabetes, increases in renal size and concentration of glucocerebroside and ganglioside GM3 were observed paralleling an increase in UDP-Glc concentration. GlcCer synthase and beta-glucosidase-specific activities were no different between control and diabetic kidneys. The apparent Km of the GlcCer synthase with respect to UDP-Glc was 250 microM and was unchanged in the diabetic kidneys. The observed concentrations of UDP-Glc were 149 and 237 microM in control and diabetic kidneys, respectively. The UDP-Glc level is thus rate limiting with regard to GlcCer synthesis. To determine whether the changes in glycolipid content were functionally significant, diabetic and control groups were treated with the GlcCer synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1- propanol, 2 wk after the induction of diabetes. Kidney weights in the diabetic rats treated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol were no different than the control groups. Morphometric analysis of glomerular volumes paralleled changes in renal growth. Glycosphingolipid formation may therefore represent a significant pathway for glucose utilization in early diabetic nephropathy.
