Isolation and Functional Analysis of Mouse UbA52 Gene and Its Relevance to Diabetic Nephropathy

Abstract

In delineating the mechanism(s) of diabetic nephropathy various novel genes have been isolated, whereas others remain to be discovered. We identified several up-regulated genes in the kidneys of diabetic newborn mice. Among them was UbA52, a ubiquitin ribosomal fusion protein. Its mRNA expression in the kidney was proportional to blood glucose levels. By in situ hybridization and immunohistochemistry, UbA52 was exclusively localized to renal tubules, and its expression was markedly increased in diabetic mice. The up-regulated UbA52 mRNA and protein expression were also observed in Madin-Darby canine kidney cells, a tubular cell line, treated with 30 mM glucose in both cell lysates and ribosomal fractions. To explore the mechanism(s) of its increased expression, UbA52 genomic DNA was isolated. A transcription start site at ؊22 bp from the initiation codon was identified and confirmed by primer extension analysis. The UbA52 promoter region included glucose response-related E-box sequences and stress response elements (STRE). Unlike in humans, mouse UbA52 gene had no introns in the coding or 5-ATG-flanking regions. To identify the DNA segment with maximal promoter activity, deletion constructs were prepared using a pSEAP vector system and transfected into COS7 kidney cells. Maximal activity was confined to ؊198 to ؉68 bp, which included E-boxes and STRE motifs. A dose-dependent increase in the promoter activity was observed in cells exposed to high glucose. Mutations in the first E-box (CAGCTG 3 TGGCTG) or STRE (CCCCT 3 CATCT) resulted in a decrease in the SEAP activity under high glucose ambience. Given the presence of glucose-responsive motifs in the promoter region and decrease in the SEAP activity in E-box mutants in the presence of glucose, these data suggest that UbA52, a ribosomal fusion protein, may be relevant in the pathogenesis of diabetic nephropathy.

Modulation of renal-specific oxidoreductase/ myo -inositol oxygenase by high-glucose ambience

“…7 A-F). Related to ChRE are the consensus sequences of E boxes recently described in promoters of various genes, e.g., UbA 52 , ubiquitin ribosomal fusion protein, and TGF-␤1, affected by highglucose ambience (36,37). When consensus sequences are used, a dose-dependent binding activity in cells exposed to D-glucose or sorbitol͞myo-inositol was observed (Fig.…”

“…8 A-D) suggests that AP1 and Sp1 modulate the transcription of this enzyme and, thereby, the pathobiology of renal tubules. In regard to STRE, AP1 and Sp1 have been described in genes containing an E box responsive to hyperglycemia, e.g., UbA 52 (36), and these promoter elements also seem to be functional in this enzyme, as indicated by DNA-protein interactions ( Fig. 8 E and F).…”

Characterization of Diabetic Nephropathy by Urinary Proteomic Analysis: Identification of a Processed Ubiquitin Form as a Differentially Excreted Protein in Diabetic Nephropathy Patients

“…Gene expression analyses displayed an overexpression of the UbA52 protein in kidney of diabetic newborn mice (36 ). UbA52 expression is regulated by various types of injuries, such as oxidative and carbonyl stress, which are important factors in the pathophysiology of diabetic nephropathy and apoptosis (36,37 ). Selective expression of UbA52 in the renal tubules suggests that the ubiquitin-proteasome proteolytic system is indeed operative in this compartment of the kidney and might play an important role in diabetic nephropathy.…”

“…In the kidney, the UbA52 was exclusively located in renal tubules and its expression in mouse kidney was found to be proportional to the glucose concentrations in blood. Gene expression analyses displayed an overexpression of the UbA52 protein in kidney of diabetic newborn mice (36 ). UbA52 expression is regulated by various types of injuries, such as oxidative and carbonyl stress, which are important factors in the pathophysiology of diabetic nephropathy and apoptosis (36,37 ).…”

Expression of Myosin Light Chain Kinase in Kidney of Streptozotocin-Induced Diabetic Rats

“…A wide variety of mechanisms in the pathogenesis of diabetes have been proposed, including accumulation of nonenzymatic glycated end products in the kidney, oxidation of renal glycoproteins by reactive oxygen species, intracellular accumulation of sorbitol generated by the reduction of glucose by aldose reductase, involvement of mitogen-activated protein kinase and growth factors [5][6][7][8]. The diverse mechanisms indicates that a vast number of molecules and different signal transduction pathways are involved in its pathogenesis [9,10]. But, the mechanism of diabetic nephropathy remains incompletely understood.…”