Diabetic kidney disease is the leading cause of ESRD, but few biomarkers of diabetic kidney disease are available. This study used gas chromatography-mass spectrometry to quantify 94 urine metabolites in screening and validation cohorts of patients with diabetes mellitus (DM) and CKD(DM+CKD), in patients with DM without CKD (DM-CKD), and in healthy controls. Compared with levels in healthy controls, 13 metabolites were significantly reduced in the DM+CKD cohorts (P#0.001), and 12 of the 13 remained significant when compared with the DM-CKD cohort. Many of the differentially expressed metabolites were water-soluble organic anions. Notably, organic anion transporter-1 (OAT1) knockout mice expressed a similar pattern of reduced levels of urinary organic acids, and human kidney tissue from patients with diabetic nephropathy demonstrated lower gene expression of OAT1 and OAT3. Analysis of bioinformatics data indicated that 12 of the 13 differentially expressed metabolites are linked to mitochondrial metabolism and suggested global suppression of mitochondrial activity in diabetic kidney disease. Supporting this analysis, human diabetic kidney sections expressed less mitochondrial protein, urine exosomes from patients with diabetes and CKD had less mitochondrial DNA, and kidney tissues from patients with diabetic kidney disease had lower gene expression of PGC1a (a master regulator of mitochondrial biogenesis). We conclude that urine metabolomics is a reliable source for biomarkers of diabetic complications, and our data suggest that renal organic ion transport and mitochondrial function are dysregulated in diabetic kidney disease.

Diabetic microvascular complications have been considered to be mediated by a glucose-driven increase in mitochondrial superoxide anion production. Here, we report that superoxide production was reduced in the kidneys of a steptozotocin-induced mouse model of type 1 diabetes, as assessed by in vivo real-time transcutaneous fluorescence, confocal microscopy, and electron paramagnetic resonance analysis. Reduction of mitochondrial biogenesis and phosphorylation of pyruvate dehydrogenase (PDH) were observed in kidneys from diabetic mice. These observations were consistent with an overall reduction of mitochondrial glucose oxidation. Activity of AMPK, the major energy-sensing enzyme, was reduced in kidneys from both diabetic mice and humans. Mitochondrial biogenesis, PDH activity, and mitochondrial complex activity were rescued by treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). AICAR treatment induced superoxide production and was linked with glomerular matrix and albuminuria reduction in the diabetic kidney. Furthermore, diabetic heterozygous superoxide dismutase 2 (Sod2 +/-) mice had no evidence of increased renal disease, and Ampka2 -/-mice had increased albuminuria that was not reduced with AICAR treatment. Reduction of mitochondrial superoxide production with rotenone was sufficient to reduce AMPK phosphorylation in mouse kidneys. Taken together, these results demonstrate that diabetic kidneys have reduced superoxide and mitochondrial biogenesis and activation of AMPK enhances superoxide production and mitochondrial function while reducing disease activity. IntroductionComplications of diabetes, including retinopathy, neuropathy, and nephropathy, are responsible for considerable morbidity, and are experienced by a majority of individuals with diabetes over time. How elevated glucose levels initiate these complications remains unclear. The prevailing theory states that enhanced mitochondrial production of superoxide anion in response to elevated cellular glucose concentrations leads to pathological pathway activation and cell dysfunction (1, 2). Studies in cell culture systems demonstrated that addition of high glucose produced an increase in ROS that has been attributed to the mitochondrial electron transport chain (ETC). The currently accepted scheme is that chronically elevated glucose leads to increased ROS production by mitochondria, contributing to downstream cellular injury processes, and ultimately resulting in end-organ dysfunction and structural changes. Based on this theory, numerous strategies are being developed to interrupt the mitochondrial production of superoxide and inhibit downstream deleterious pathways.

The most prevalent DNA lesions induced by UVB are the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). It has been a long standing controversy as to which of these photoproduct is responsible for mutations in mammalian cells. Here we have introduced photoproduct-specific DNA photolyases into a mouse cell line carrying the transgenic mutation reporter genes lacI and cII. Exposure of the photolyase-expressing cell lines to photoreactivating light resulted in almost complete repair of either CPDs or (6-4)PPs within less than 3 h. The mutations produced by the remaining, nonrepaired photoproducts were scored. The mutant frequency in the cII gene after photoreactivation by CPD photolyase was reduced from 127 ؋ 10 ؊5 to 34 ؋ 10 ؊5 (background, 8 -10 ؋ 10 ؊5 ). Photoreactivation with (6-4) photolyase did not lower the mutant frequency appreciably. In the lacI gene the mutant frequency after photoreactivation repair of CPDs was reduced from 148 ؋ 10 ؊5 to 28 ؋ 10 ؊5(background, 6 -10 ؋ 10 ؊5 ). Mutation spectra obtained with and without photoreactivation by CPD photolyase indicated that the remaining mutations were derived from background mutations, unrepaired CPDs, and other DNA photopoducts including perhaps a small contribution from (6-4)PPs. We conclude that CPDs are responsible for at least 80% of the UVB-induced mutations in this mammalian cell model.The UV component of sunlight is responsible for the induction of skin tumors, most notably basal cell and squamous cell carcinomas and probably also melanomas (1, 2). Mutations in the p53 gene have been found in a large percentage of human skin malignancies (3-6). The most frequent p53 mutations in skin tumors are C to T or CC to TT mutations involving dipyrimidine sequences. These are considered characteristic mutational changes that can be ascribed to solar UV irradiation (7).The most abundant UV-induced DNA photoproducts are the cis-syn cyclobutane pyrimidine dimers (CPDs) 1 and the pyrimidine (6-4) pyrimidone photoproducts ((6-4)PPs). Both lesions are produced by UVB (280 -320 nm) and UVC (200 -280 nm) irradiation in DNA (8, 9). Most of the mutagenic and carcinogenic action of sunlight has been ascribed to the UVB portion of the solar spectrum (10) with a possible role for UVA (320 -400 nm) in the induction of melanoma (11). The absorption of UVA photons by DNA is rather weak, and it is thought that indirect damaging mechanisms may involve endogenous chromophores as radiation absorbing intermediates (12). These can generate reactive oxygen species, which may then damage DNA. Of all lesions formed in DNA after UVB irradiation, the CPD is considered one of the most important ones based on its relatively high abundance, slow repair, and known mutagenicity (8, 9, 13). However, a strong case can be made that the (6-4)PPs are equally if not more important than the CPDs for inducing mutations. C to T transitions can be induced by both CPDs and (6-4)PPs in mutagenesis studies using site-specific photolesions, and (6-4)PP...

The prevalence of diabetic nephropathy continues to rise, highlighting the importance of investigating and discovering novel treatment strategies. TRB3 is a kinase-like molecule that modifies cellular survival and metabolism and interferes with signal transduction pathways. Herein, we report that TRB3 expression is increased in the kidneys of type 1 and type 2 diabetic mice. TRB3 is expressed in conditionally immortalized podocytes; however, it is not stimulated by elevated glucose. The diabetic milieu is associated with increased oxidative stress and circulating free fatty acids (FFA). We show that reactive oxygen species (ROS) such as H(2)O(2) and superoxide anion (via the xanthine/xanthine oxidase reaction) as well as the FFA palmitate augment TRB3 expression in podocytes. C/EBP homologous protein (CHOP) is a transcription factor that is associated with the endoplasmic reticulum stress response. CHOP expression increases in diabetic mouse kidneys and in podocytes treated with ROS and FFA. In podocytes, transfection of CHOP increases TRB3 expression, and ROS augment recruitment of CHOP to the proximal TRB3 promoter. MCP-1/CCL2 is a chemokine that contributes to the inflammatory injury associated with diabetic nephropathy. In these studies, we demonstrate that TRB3 can inhibit basal and stimulated podocyte production of MCP-1. In summary, enhanced ROS and/or FFA associated with the diabetic milieu induce podocyte CHOP and TRB3 expression. Because TRB3 inhibits MCP-1, manipulation of TRB3 expression could provide a novel therapeutic approach in diabetic kidney disease.