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      Renal Gene Expression in Embryonic and Newborn Diabetic Mice

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          Several novel genes that are upregulated in diabetic kidneys have been identified. Recently, transforming growth factor beta driven secreted proteins, i.e., connective tissue growth factor and gremlin (bone morphogenetic protein 2), have been identified, and their expression has been correlated with the tissue changes seen in diabetic nephropathy in the adult population. However, there are very few studies reported in the literature that describe the gene expression in the diabetic state during embryonic and neonatal life. It is well known that exposure to glucose or its epimer, i.e., mannose, induces marked dysmorphogenesis of the embryonic metanephros in an organ culture system. These changes are associated with ATP depletion and marked apoptosis, suggesting an oxidant stress in the induction of dysmorphogenesis of the embryonic metanephros. In view of the glucose-induced changes in the fetal metanephros, a diabetic state was induced by the administration of streptozotocin during pregnancy, and newborn mouse kidneys were processed for suppression subtractive hybridization-PCR. In addition, a diabetic state was induced in newborn diabetic mice, and after 1 week their kidneys were harvested and subjected to representational difference analysais of cDNA. Four novel genes with upregulated mRNA expression were identified. They included: (1) a translocase inner mitochondrial membrane 44 that is involved in the ATP-dependent import of preproteins from the cytosol into the mitochondrial matrix; (2) a kidney-specific aldo-keto reductase that utilizes NADPH and NADH as cofactors in the reduction of aromatic aldehydes and aldohexoses; (3) Rap1b, a Ras-related small GTP-binding protein that behaves as a GTPase and cycles between GTP-bound (active) and GDP-bound (inactive) states associated with conformational change, and (4) a fusion protein of ubiquitin polypeptide and ribosomal protein L40 (UbA<sub>52</sub> or ubiquitin/60) that is intimately involved in the ubiquitin-dependent proteasome pathway related to the accelerated degradation of proteins under various stress conditions, such as those seen in patients with cancer and diabetes mellitus.

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          Most cited references 4

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          Substrate targeting in the ubiquitin system.

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            Characterization of mammalian translocase of inner mitochondrial membrane (Tim44) isolated from diabetic newborn mouse kidney.

             J Wada,  Y Kanwar (1998)
            Mammalian translocase of mitochondrial inner membrane (mTim44) was isolated during representational difference analysis of cDNA from diabetic mouse kidney. Streptozotocin-induced diabetic mouse kidney cDNA was prepared and subtracted by normal mouse kidney cDNA. By using one of the isolated cDNA fragments as a screening probe, full-length cDNA of mTim44 was isolated from lambdaZAP kidney cDNA library. At the nucleotide level, mTim44 did not exhibit significant homology with any known genes; however, at the amino acid level, it had 50% similarity and 29% identity with yeast Tim44. C-terminal FLAG epitope-tagged mTim44 fusion protein was transiently expressed in COS7 cells. By using anti-FLAG epitope M2 monoclonal antibody, mTim44 was found to have its subcellular localization associated with mitochondria. By immunoelectron microscopy, mTim44 was seen in the paracrystalline structures within the mitochondria, as well as in their cristae. Mitochondrial import assay of in vitro translated mTim44 indicated that its precursor product ( approximately 50 kDa) was imported and proteolytically processed to a mature approximately 44-kDa protein, and its translocation was inner membrane potential (DeltaPsi)-dependent. Imported mTim44 was protected from protease digestion in which outer membranes were selectively permeabilized with digitonin. The mature form of mTim44 could be recovered in the supernatant of sonicated mitochondrial membrane fraction treated with 0.1 M Na2CO3, pH 11.5. The data indicate that mTim44 is a mitochondrial inner membrane protein, one of the members of the mammalian TIM complex and up-regulated in hyperglycemic states.
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              Identification of a renal-specific oxido-reductase in newborn diabetic mice.

               Y Kanwar,  Q. Yang,  B. Dixit (2000)
              Aldose reductase (ALR2), a NADPH-dependent aldo-keto reductase (AKR), is widely distributed in mammalian tissues and has been implicated in complications of diabetes, including diabetic nephropathy. To identify a renal-specific reductase belonging to the AKR family, representational difference analyses of cDNA from diabetic mouse kidney were performed. A full-length cDNA with an ORF of 855 nt and yielding a approximately 1.5-kb mRNA transcript was isolated from a mouse kidney library. Human and rat homologues also were isolated, and they had approximately 91% and approximately 97% amino acid identity with mouse protein. In vitro translation of the cDNA yielded a protein product of approximately 33 kDa. Northern and Western blot analyses, using the cDNA and antirecombinant protein antibody, revealed its expression exclusively confined to the kidney. Like ALR2, the expression was up-regulated in diabetic kidneys. Its mRNA and protein expression was restricted to renal proximal tubules. The gene neither codistributed with Tamm-Horsfall protein nor aquaporin-2. The deduced protein sequence revealed an AKR-3 motif located near the N terminus, unlike the other AKR family members where it is confined to the C terminus. Fluorescence quenching and reactive blue agarose chromatography studies revealed that it binds to NADPH with high affinity (K(dNADPH) = 66.9 +/- 2.3 nM). This binding domain is a tetrapeptide (Met-Ala-Lys-Ser) located within the AKR-3 motif that is similar to the other AKR members. The identified protein is designated as RSOR because it is renal-specific with properties of an oxido-reductase, and like ALR2 it may be relevant in the renal complications of diabetes mellitus.

                Author and article information

                Nephron Exp Nephrol
                Cardiorenal Medicine
                S. Karger AG
                05 April 2002
                : 10
                : 2
                : 130-138
                Departments of aMedicine and bPathology, Northwestern University Medical School, Chicago, Ill., USA; cThird Department of Medicine, Okayama University, Okayama, Japan
                49908 Exp Nephrol 2002;10:130–138
                © 2002 S. Karger AG, Basel

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                Page count
                Figures: 7, References: 40, Pages: 9
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/49908

                Cardiovascular Medicine, Nephrology

                Diabetes mellitus, Renal development, Gene expression


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