Malou Friederich

Diabetes, Oxidative Stress, Nitric Oxide and Mitochondria Function

The role of altered mitochondria function has recently emerged as an important mechanism for the development of diabetic complications. Altered mitochondria function has also been implicated in the ageing process, defective insulin secretion, hypertension, arteriosclerosis, ischemia-reperfusion injury and apoptosis. Normally, the mitochondria are associated with ATP production using primarily pyruvate as the substrate, but recent reports indicate that tissue specific preferences exist. Also, the mitochondria are a substantial source of superoxide production, preferentially during states of elevated intracellular glucose concentrations. The mitochondria function is regulated by several factors including nitric oxide, oxidative stress, mammalian target of rapamycin, ADP and P(i) availability, which result in a complex regulation of ATP production and oxygen consumption, but also superoxide generation. These factors seem to be tissue specific, which warrants a more diverse mechanistic model applying to that specific tissue or cell type. This review presents the basic functions of the mitochondria and focuses on the complex interplay between oxidative stress, nitric oxide and uncoupling proteins in regulating mitochondria function with special focus on diabetes-induced alterations occurring on the mitochondria level.

Diabetes-induced up-regulation of uncoupling protein-2 results in increased mitochondrial uncoupling in kidney proximal tubular cells

We have previously reported increased O(2) consumption unrelated to active transport by tubular cells and up-regulated mitochondrial uncoupling protein (UCP)-2 expressions in diabetic kidneys. It is presently unknown if the increased UCP-2 levels in the diabetic kidney results in mitochondrial uncoupling and increased O(2) consumption, which we therefore investigated in this study. The presence of UCP-2 in proximal tubular cells was confirmed by immunohistochemistry and found to be increased (western blot) in homogenized tissue and isolated mitochondria from kidney cortex of diabetic rats. Isolated proximal tubular cells had increased total and ouabain-insensitive O(2) consumption compared to controls. Isolated mitochondria from diabetic animals displayed increased glutamate-stimulated O(2) consumption (in the absence of ADP and during inhibition of the ATP-synthase by oligomycin) compared to controls. Guanosine diphosphate, an UCP inhibitor, and bovine serum albumin which removes fatty acids that are essential for UCP-2 uncoupling activity, independently prevented the increased glutamate-stimulated O(2) consumption in mitochondria from diabetic animals. In conclusion, diabetic rats have increased mitochondrial UCP-2 expression in renal proximal tubular cells, which results in mitochondrial uncoupling and increased O(2) consumption. This mechanism may be protective against diabetes-induced oxidative stress, but will increase O(2) usage. The subsequently reduced O(2) availability may contribute to diabetes-induced progressive kidney damage.

Identification and distribution of uncoupling protein isoforms in the normal and diabetic rat kidney

Uncoupling protein (UCP)-2 and -3 are ubiquitously expressed throughout the body but there is currently no information regarding the expression and distribution of the different UCP isoforms in the kidney. Due to the known cross-reactivity of the antibodies presently available for detection of UCP-2 and -3 proteins, we measured the mRNA expression of UCP-1, -2 and -3 in the rat kidney in order to detect the kidney-specific UCP isoforms. Thereafter, we determined the intrarenal distribution of the detected UCP isoforms using immunohistochemistry. Thereafter, we compared the protein levels in control and streptozotocin-induced diabetic rats using Western blot. Expressions of the UCP isoforms were also performed in brown adipose tissue and heart as positive controls for UCP-1 and 3, respectively. UCP-2 mRNA was the only isoform detected in the kidney. UCP-2 protein expression in the kidney cortex was localized to proximal tubular cells, but not glomerulus or distal nephron. In the medulla, UCP-2 was localized to cells of the medullary thick ascending loop of Henle, but not to the vasculature or parts of the nephron located in the inner medulla. Western blot showed that diabetic kidneys have about 2.5-fold higher UCP-2 levels compared to controls. In conclusion, UCP-2 is the only isoform detectable in the kidney and UCP-2 protein can be detected in proximal tubular cells and cells of the medullary thick ascending loop of Henle. Furthermore, diabetic rats have increased UCP-2 levels compared to controls, but the mechanisms underlying this increase and its consequences warrants further studies.

Uncoupling protein-2 in diabetic kidneys: increased protein expression correlates to increased non-transport related oxygen consumption.

Diabetic patients have an elevated risk to develop renal dysfunction and it has been postulated that altered energy metabolism is involved. We have previously shown that diabetic rats have markedly decreased oxygen availability in the kidney, resulting from increased oxygen consumption. A substantial part of the increased oxygen consumption is unrelated to tubular transport, suggesting decreased mitochondrial efficiency. In this study, we investigated the protein expression of mitochondrial uncoupling protein (UCP)-2 in kidney tissue from control and streptozotocin (STZ)-induced diabetic rats. Protein levels of UCP-2 were measured in adult male control and STZ-diabetic Wistar Furth as well as Sprague Dawley rats in both the kidney cortex and medulla by Western blot technique. Two weeks of hyperglycemia resulted in increased protein levels of UCP-2 in kidneys from both Wistar Furth and Sprague Dawley rats. Both cortical and medullary UCP-2 levels were elevated 2-3 fold above control levels. We conclude that sustained STZ-induced hyperglycemia increases the kidney levels of mitochondrial UCP-2, which could explain the previously reported increase in non-transport related oxygen consumption in diabetic kidneys. The elevated UCP-2 levels may represent an effort to reduce the increased production of superoxide radicals which is evident during diabetes.

Early transcriptional regulation by C-peptide in freshly isolated rat proximal tubular cells

Clinical studies have shown that proinsulin C‐peptide exerts renoprotective effects in type 1 diabetes, although the underlying mechanisms are poorly understood. As C‐peptide has been shown to induce several intracellular events and to localize to nuclei, we aimed to determine whether gene transcription is affected in proximal tubular kidney cells, and if so, whether the genes with altered transcription include those related to protective mechanisms.