Mitochondrial Glutathione in Diabetic Nephropathy

Abstract

Although there are many etiologies for diabetic nephropathy (DN), one common characteristic of all cases involves mitochondrial oxidative stress and consequent bioenergetic dysfunction. As the predominant low-molecular-weight, intramitochondrial thiol reductant, the mitochondrial glutathione (mtGSH) pool plays important roles in how this organelle adapts to the chronic hyperglycemia and redox imbalances associated with DN. This review will summarize information about the processes by which this important GSH pool is regulated and how manipulation of these processes can affect mitochondrial and cellular function in the renal proximal tubule. Mitochondria in renal proximal tubular (PT) cells do not appear to synthesize GSH de novo but obtain it by transport from the cytoplasm. Two inner membrane organic anion carriers, the dicarboxylate carrier (DIC; Slc25a10) and 2-oxoglutarate carrier (OGC; Slc25a11) are responsible for this transport. Genetic modulation of DIC or OGC expression in vitro in PT cells from diabetic rats can alter mitochondrial function and susceptibility of renal PT cells to oxidants, with overexpression leading to reversion of bioenergetic conditions to a non-diabetic state and protection of cells from injury. These findings support the mtGSH carriers as potential therapeutic targets to correct the underlying metabolic disturbance in DN.

Keywords: diabetic nephropathy; gene expression; glutathione; mitochondria; oxidative stress; transport.

Figure 1

General scheme of major events leading from chronic hyperglycemia to mitochondrial dysfunction. Abbreviations: GSH, glutathione; RNS, reactive nitrogen species; ROS, reactive oxygen species.

Figure 2

Pathways of glutathione transport across the mitochondrial inner membrane. Minus signs after each arrow indicate inhibition of the transporter activity. Abbreviations: DIC, dicarboxylate carrier; GS²⁻, glutathione; OGC, 2-oxoglutarate carrier; P_i²⁻, inorganic phosphate.

Figure 3

Summary of selected data on mitochondrial glutathione status in kidneys of diabetic rats. Data are summarized from a previously published study [50] and are shown without error bars for simplicity. Values are means of measurements from 4 control and 4 diabetic rats for panel (A) and (B). For panel (C), PCR values are means of measurements from 9 and 12 control and 9 and 12 diabetic rats for 1-month and 3-month studies, respectively; Western blot values are means of measurements from 3 control and 3 diabetic rats. (A) Mitochondrial GSH (mtGSH) concentrations were determined with the GSH-Glo^TM chemiluminescence kit from Promega (Madison, WI, USA). (B) mtGSH transport into isolated mitochondria was measured with ³⁵S-labeled GSH as described in reference [50]. (C) Real-time quantitative PCR measurements of DIC and OGC mRNA in renal cortical samples were done as described in reference [50], using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control and the ∆C_T method for determination of relative expression levels. (D) Western blots were performed by enhanced chemiluminescence with rabbit anti-rat polyclonal antibodies to the DIC and OGC. Expression levels were normalized to that of the voltage-dependent anion channel (VDAC) and band density was derived using GelEval 1.3.7 software for Mac OS X.

Figure 4

Scheme of functional and biochemical responses to diabetes or uninephrectomy that are associated with changes in mitochondrial redox status. Abbreviations: GFR, glomerular filtration rate; GSH, glutathione; RBF, renal blood flow; ROS, reactive oxygen species. The scheme is based on one published in reference [25] with some modifications.

Figure 5

Pathways of glutathione transport in renal proximal tubular cells. Shown are the major pathways for glutathione (GSH) across both plasma membranes and the mitochondrial inner membrane (MIM). The critical importance of mitochondrial energetics in determining both mitochondrial and overall cellular GSH status is apparent from the integration of transport pathways with the citric acid cycle and ATP generation. The figure is based on one published in reference [25] with slight modifications. Abbreviations: BBM, brush-border membrane; BLM, basolateral membrane; DIC, dicarboxylate carrier; Mrp2/4, multidrug resistance proteins 2/4; NaC3, sodium-dicarboxylate carrier 3; OA, organic anion; Oat1/3, organic anion transporter 1/3; Oatp1a1; organic anion transporting polypeptide 1a1; 2-OG, 2-oxoglutarate; OGC, oxoglutarate carrier; P_i²⁻, inorganic phosphate.