Adaptive changes in renal mitochondrial redox status in diabetic nephropathy

Toxicology and Applied Pharmacology 258 (2012) 188–198

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Adaptive changes in renal mitochondrial redox status in diabetic nephropathy David A. Putt, Qing Zhong, Lawrence H. Lash ⁎ Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA

a r t i c l e

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Article history: Received 24 August 2011 Revised 18 October 2011 Accepted 18 October 2011 Available online 7 November 2011 Keywords: Rat kidney Diabetes Oxidative stress Mitochondrial function Glutathione Dicarboxylate transport

a b s t r a c t Nephropathy is a serious and common complication of diabetes. In the streptozotocin (STZ)-treated rat model of diabetes, nephropathy does not typically develop until 30 to 45 days post-injection, although hyperglycemia occurs within 24 h. We tested the hypothesis that chronic hyperglycemia results in a modest degree of oxidative stress that is accompanied by compensatory changes in certain antioxidants and mitochondrial redox status. We propose that as kidneys progress to a state of diabetic nephropathy, further adaptations occur in mitochondrial redox status. Basic parameters of renal function in vivo and several parameters of mitochondrial function and glutathione (GSH) and redox status in isolated renal cortical mitochondria from STZ-treated and age-matched control rats were examined at 30 days and 90 days post-injection. While there was no effect of diabetes on blood urea nitrogen, measurement of other, more sensitive parameters, such as urinary albumin and protein, and histopathology showed significant and progressive worsening in diabetic rats. Thus, renal function is compromised even prior to the onset of frank nephropathy. Changes in mitochondrial respiration and enzyme activities indicated existence of a hypermetabolic state. Higher mitochondrial GSH content and rates of GSH transport into mitochondria in kidneys from diabetic rats were only partially due to changes in expression of mitochondrial GSH carriers and were mostly due to higher substrate supply. Although there are few clear indicators of oxidative stress, there are several redox changes that occur early and change further as nephropathy progresses, highlighting the complexity of the disease. © 2011 Elsevier Inc. All rights reserved.

Introduction Renal complications from both Type 1 and Type 2 diabetes occur in up to 40% of all patients and are a frequent cause of death (Geiss et al., 1993; Ibrahim and Hostetter, 1997; Sedor, 2006; Susztak and Böttinger, 2006). The increasing incidence of diabetes in the U.S. indicates that prevention and treatment of diabetic renal disease are a critical public health issue and that an improved understanding of the underlying biochemical mechanisms of diabetic nephropathy is likewise critical in the development of novel therapeutic approaches (Remuzzi et al., 2006).

Abbreviations: Acivicin, L-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; BG, blood glucose; BW, body weight; BUN, blood urea nitrogen; DIC, dicarboxylate carrier; DMEM:F12, Dulbecco's Modified Eagle's Medium:Ham's F12; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GCS, γ-glutamylcysteine synthetase; GDH, glutamate dehydrogenase; GGT, γ-glutamyltransferase; GPX, glutathione peroxidase; GRD, glutathione disulfide reductase; GSH, glutathione; GST, glutathione S-transferase; H&E, hematoxylin and eosin; HNE, 4-hydroxy-2-nonenal; KW, kidney weight; MDH, malate dehydrogenase; MVK, methyl vinyl ketone; NAG, N-acetyl-β-D-glucosaminidase; Oat3, organic anion transporter 3; 2-OG, 2-oxoglutarate; OGC, 2-oxoglutarate carrier; PAS, periodic acid-Schiff base; PT, proximal tubular; RCR, respiratory control ratio; ROS, reactive oxygen species; SDH, succinate dehydrogenase; SOD2, superoxide dismutase 2; STZ, streptozotocin; tBH, tert-butyl hydroperoxide; Trx2, thioredoxin 2; TTBS, Tris-buffered saline containing Tween 20; U-alb, urinary albumin; U-prot, urinary protein; VDAC, voltage-dependent anion channel. ⁎ Corresponding author at: Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA. Fax: +1 313 577 6739. E-mail address: [email protected] (L.H. Lash). 0041-008X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2011.10.021

Although glycemic control is certainly critical for long-term preservation of renal function (Fioretto et al., 2006; Remuzzi et al., 2006), there is a large amount of evidence in the literature that suggests the underlying mechanism of renal cellular damage in diabetic nephropathy involves various manifestations of oxidative and nitrosative stress (Alderson et al., 2004; Beisswenger et al., 2005; Brownlee, 2001; Chander et al., 2004; Hakim and Pflueger, 2010; Obrosova et al., 2003; Prabhakar et al., 2007). Depletion of renal glutathione (GSH)1 content has been demonstrated in various models of diabetic nephropathy (Beisswenger et al., 2005; Brownlee, 2001; Chander et al., 2004; Obrosova et al., 2003; Winiarska et al., 2004) and dietary supplementation with GSH can be protective against some of the pathologies associated with diabetes, including diabetic nephropathy (Ueno et al., 2002). These observations suggest that in diabetic nephropathy, cellular GSH status is either a critical target or a determinant of the underlying pathological processes occurring in the renal proximal tubule. A basic concept has emerged in recent years that mitochondrial dysfunction, often including redox disturbances, plays a fundamental and underlying role in many metabolic and degenerative diseases, including diabetes, cancer, and aging (Wallace, 2005). Indeed, alterations of mitochondrial function have been strongly implicated as a critical early step in the development of renal tubular cell damage that results from diabetes (Rolo and Palmeira, 2006). Function of the dicarboxylate carrier (DIC; Slc25a10) and 2-oxoglutarate carrier (OGC; Slc25a11) as the primary mitochondrial inner membrane carrier proteins that determine the GSH pool in the mitochondrial matrix (Chen and Lash, 1998; Chen

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et al., 2000; McKernan et al., 1991) suggests that alterations in substrate supply due to changes in mitochondrial intermediary metabolism will impact GSH status. Hence, energetics and redox status in renal mitochondria are likely to be linked. Thus, although there has been considerable work on different aspects of renal GSH status in diabetes, very little is known about the mitochondrial GSH pool in diabetes and no work has been done on effects of specifically targeting this pool to develop a novel therapeutic approach. Previous work of ours (Zhong and Lash, 2007) showed some preliminary studies of mitochondrial and redox status in renal subcellular fractions from kidneys of rats made diabetic by an injection of streptozotocin (STZ). The studies were conducted at 30 days after injection of STZ, which is a time point at which rats are clearly hyperglycemic but are not believed to exhibit frank nephropathy. The results demonstrated that mitochondrial respiratory activity and citric acid cycle enzymes are increased in kidneys of 1-month diabetic rats. Somewhat surprisingly, total cellular and mitochondrial GSH concentrations were significantly higher in kidneys of diabetic rats as well. The higher GSH concentrations were interpreted as being a compensatory response to the enhanced mitochondrial activity and accompanying oxidative stress. Besides those preliminary ex vivo studies, we recently characterized mitochondrial function, redox status, and GSH status in primary cultures of proximal tubular (PT) cells from 1-month STZ-diabetic and age-matched control rats (Zhong et al., 2009). These studies confirmed the increased mitochondrial activity, basal oxidative stress, and mitochondrial GSH levels in PT cells from diabetic rats. They further demonstrated that the PT cells from diabetic rats were more susceptible to acute cytotoxicity from oxidants, thiol alkylating agents, and a mitochondrial inhibitor. These data showing increased cytotoxicity in PT cells from diabetic rats as compared to those from age-matched control rats suggest that diabetes should be considered an additional risk factor in the development of chemically induced nephrotoxicity. The goals of the present study were to extend our previous work to more fully characterize mitochondrial function and GSH status in 1-month STZ-diabetic rat kidneys and to compare these processes in kidneys of 3-month STZ-diabetic rats. We will first validate and extend the hypothesis that in 1-month STZ-diabetic rats, mitochondrial activity is increased, leading to compensatory increases in mitochondrial GSH contents. Further, we hypothesize that as diabetic nephropathy progresses, some adaptation will occur in mitochondrial function and GSH status, although these changes will be insufficient to prevent oxidative

Fig. 1. Summary scheme illustrating demonstrated and proposed relationships between hyperglycemia, the development of oxidative stress, alterations in mitochondrial glutathione status, and toxicological implications.

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stress and nephropathy. These hypotheses are illustrated in the scheme in Fig. 1. The critical, priming event in the pathology is the increase in renal mitochondrial oxygen consumption that results from the chronic hyperglycemia. This in turn subsequently leads to increased formation and release of reactive oxygen species (ROS). In response to the higher levels of ROS and the hypermetabolic state in renal mitochondria, we propose that a variety of antioxidant enzymes and substrate transporters are increased in expression and/or activity. Despite the increased level of some antioxidants or increased activity of some antioxidant enzymes, the renal PT cell is stressed so that susceptibility to various classes of toxicants is increased. Materials and methods Experimental design. Effects of chronic hyperglycemia and diabetes on the renal PT cell, and specifically mitochondria within those cells, were investigated at both the in vivo and in vitro levels. Animals were studied at two time points relative to the onset of diabetes, 30 days and 90 days after receiving an STZ injection to make them diabetic. The significance of these time points is that the earlier one is at a time prior to development of significant signs of overt nephropathy whereas the latter one is at a time after the typical onset of nephropathy in this model (Tesch and Allen, 2007). Although additional time points post-STZ injection (e.g., 60 days and 120 days) were originally included in the experimental plan, preliminary measurements showed only modest differences between responses at 30 vs. 60 days and 90 vs. 120 days. Accordingly, more extensive studies were conducted and data are reported only for 30- and 90-day diabetic and age-matched control rats. Chemicals and materials. L-[ 3H-glycyl]-GSH (44.8 Ci/mmol) was purchased from PerkinElmer (Waltham, MA). [2- 14C]-Malonate (56 mCi/mmol) was purchased from ICN Biochemicals (Irvine, CA). [ 14C]-2-oxoglutarate (2-OG; 281 mCi/mmol) was purchased from PerkinElmer (Waltham, MA). Acivicin [L-(αS,5S)-α-amino-3-chloro4,5-dihydro-5-isoxazoleacetic acid] was purchased from Sigma Chemical Co. (St. Louis, MO). Other chemicals were of the highest purity available and were obtained from commercial sources. Animals and establishment of the diabetic state. Male Sprague– Dawley rats (150–174 g) were purchased from Harlan (Indianapolis, IN). All procedures with rats were conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology, as adopted by the Society of Toxicology in 1989, and all applicable federal and state regulations, and were approved by the Institutional Animal Care and Use Committee at Wayne State University. After an acclimation period of 5 days, rats were made diabetic by an ip injection of STZ (60 mg/kg body weight). Rats developed polyuria and hyperglycemia within 24–48 h. A blood glucose level >250 mg/dl was considered indicative of the diabetic state. Rats with blood glucose >550 mg/dl were given daily injections of insulin (Humulin; 1–2 units IH; Eli Lilly Co., Indianapolis, IN) to maintain them in a ketoacidosis-free, but hyperglycemic state. Diabetic and age-matched control rats were maintained for 1 month or 3 months prior to harvesting of kidneys for preparation of renal cortical homogenates and mitochondrial fractions. These STZ-treated rats are heretofore referred to as 1-month and 3-month diabetic rats, respectively. Measurement of urinary parameters. Rats were singly housed in metabolic cages for collection of 24-h urine samples for analysis of urinary albumin (U-alb), urinary protein (U-prot), and urinary activity of N-acetyl-β-D-glucosaminidase (NAG). Urinary NAG activity was measured with a kit according to the manufacturer's directions (Diazyme Laboratories, Poway, CA). Blood was collected by heart puncture at the end of the urine collection period. Serum was then isolated for

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analysis of blood urea nitrogen (BUN) levels with a BUN ELISA kit from Catachem Inc. (Stamford, CT). Preparation of isolated mitochondria. Rats were anesthetized with an ip injection of sodium pentobarbital (50 mg/kg body weight). After removal from the abdomen, rat kidneys were immediately placed in ice-cold buffer and rats were killed by bilateral pneumothorax and exsanguination. The mitochondrial isolation buffer contained 20 mM triethanolamine/HCl, pH 7.4, 225 mM sucrose, 3 mM potassium phosphate (pH 7.4), 5 mM MgCl2, 20 mM KCl, and 0.1 mM phenylmethylsulfonyl fluoride to inhibit proteolysis. EGTA (2 mM) was included in the buffer at all preparatory stages, except the final resuspension, to remove calcium ions. After decapsulation, the cortex and outer stripe of the outer medulla were sliced into small pieces and homogenized with a hand-held Dounce homogenizer. Mitochondria from homogenates of rat renal cortex were isolated by the differential centrifugation method of Johnson and Lardy (1967), as described previously (Lash and Sall, 1993). Kidneys were homogenized in 15 ml of cold buffer and centrifuged in 50-ml polycarbonate centrifuge tubes at 600 ×g (2250 rpm) for 10 min in a Sorvall SS34 rotor in a Sorvall RC2B centrifuge. Supernatant was decanted and saved. The pellets containing tissue fragments and mitochondria were washed with 30 ml of buffer and the resuspended material was centrifuged at 600 ×g for 10 min. The supernatant fractions were combined and centrifuged at 15,000 ×g for 5 min. The resulting pellet (= mitochondrial fraction) was resuspended in 2 ml of buffer without EGTA. Purity of the mitochondrial fraction obtained by this method (>90%) has been estimated by measurements of marker enzymes for potential contaminating subcellular fractions, including cytoplasm (marker = lactate dehydrogenase), endoplasmic reticulum (marker = glucose 6-phosphatase), plasma membranes (markers = γ-glutamyltransferase (GGT) and [Na ++K +]-stimulated ATPase), and lysosomes (marker = acid phosphatase) (Lash and Sall, 1993). Renal histology. Kidneys were harvested and 4-μm thick, paraffinembedded kidney sections were deparaffinized with xylene and then rehydrated through a descending gradient of ethanol. Histological morphology was examined after staining with hematoxylin and eosin (H&E) or periodic acid-Schiff base (PAS). Light microscopy was performed with a Nikon TM S microscope and images were obtained with an attached Olympus DP 12 digital camera. Measurement of mitochondrial respiratory function. After incubating mitochondria in buffer at 25 °C for 30 min, oxygen consumption in mitochondrial suspensions was measured with a Gilson 5/6H oxygraph in a thermostated, air-tight, 1.6 ml chamber at 25 °C using a Clark-type oxygen electrode. State 3 rates were measured by addition of 3.3 mM succinate in the presence of 5 μM rotenone in ethanol (final concentration = 0.3%, v/v) and 0.3 mM ADP to 1.5 to 3.0 mg mitochondrial protein; State 4 rates were measured as the rate of oxygen consumption after exhaustion of ADP. Respiratory control ratios (RCR = State 3 rate/State 4 rate) were used to assess functional integrity (Estabrook, 1967). Enzyme assays. Glutamate dehydrogenase (GDH; EC 1.4.1.2) activity was measured as NADH oxidation in the presence of 2-OG as described by Schmidt and Schmidt (1983). Malate dehydrogenase (MDH; EC 1.1.1.37) activity was measured by coupling to NADH oxidation and the consequent decrease in absorbance at 340 nm (ε = 6220 M − 1 cm − 1) (Ochoa, 1955). Succinate:cytochrome c oxidoreductase (succinate dehydrogenase, SDH) (EC 1.3.99.1) activity was measured by coupling succinate oxidation to ferricytochrome c reduction, and activity was quantitated by determining the increase in absorbance at 550.5 nm (ε = 18,500 M − 1 cm − 1) (Fleischer and

Fleischer, 1967). GGT (EC 2.3.2.2) activity was measured at 410 nm as p-nitroanilide formation with γ-glutamyl-p-nitroanilide and glycylglycine as substrates according to Orlowski and Meister (1963). γ-Glutamylcysteine synthetase (GCS; EC 6.3.2.2) activity was measured spectrophotometrically as NADPH oxidation in the presence of L-glutamate, ATP, phosphoenolpyruvate and L-aminobutyrate as substrates according to Seelig and Meister (1984). Glutathione peroxidase (GPX; EC 1.11.1.19) activity was measured spectrophotometrically as the oxidation of NADPH in the presence of GSH, hydrogen peroxide and glutathione reductase (Lawrence and Burk, 1976). Glutathione S-transferase (GST; 2.5.1.18) activity was measured spectrophotometrically at 340 nm as S-2,4-dinitrophenyl-GSH formation with 1-chloro-2,4-dinitrobenzene and GSH as substrates (Habig et al., 1974). Protein was measured using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) at a wavelength of 562 nm. A standard curve plot of bovine serum albumin concentration (0.1–0.6 mg/ml) versus absorbance was generated to determine sample protein concentration. For urinary protein measurements, urine samples were dialyzed overnight to remove uric acid. Measurement of GSH contents. GSH contents in renal homogenates and mitochondria were measured with the GSH-Glo™ kit from Promega (Madison, WI) and were quantified by chemiluminescence in a SpectraMax 2 plate reader using GSH as a standard. Measurement of GSH, malonate, and 2-OG uptake in isolated mitochondria. Before incubation with GSH, mitochondria were pretreated with 0.25 mM acivicin in the presence of 5 mM dithiothreitol for 15 min at 25 °C to inhibit the activity of GGT that derived from contaminating brush-border membranes. Pretreatment with 0.25 mM acivicin produced greater than 98% inhibition of GGT activity, both in the presence or absence of dithiothreitol (Lash and Putt, 1999). Uptake of GSH or malonate into mitochondria was determined by a single-step centrifugation method (Chen and Lash, 1998). The isolation buffer (without EGTA) was used in all incubations for transport measurements. Briefly, aliquots (0.5 ml) of samples at each time point during incubations with 0.025 μCi of radiolabeled GSH or malonate were placed in 1.5-ml polyethylene microcentrifuge tubes and were centrifuged at 13,000 g × 30 s to separate extramitochondrial space (supernatants) from mitochondrial matrix space (pellets). Initial rates of uptake were calculated from uptake time courses (0, 1, 5, 10, and 20 min) by curve-fitting and the following method. To calculate the first-order rate constant k of GSH or malonate uptake, linear curvefitting was performed on the plot of ln[Ptotal / (Ptotal − Pt)] vs. time according to Halestrap (1975). Ptotal represents the total uptake of GSH or malonate at equilibrium, which was estimated by performing an exponential decay curve-fitting on the time course data of GSH or malonate uptake. Pt represents substrate uptake at time t. Thus, the initial rate of substrate uptake was determined from the first-order rate equation v = k (Ptotal). Real-time quantitative PCR (qPCR). Total RNA was extracted from renal cortical tissues using Trizol® (Invitrogen, Carlsbad, CA) and the cDNAs were synthesized using a multiscribe reverse transcriptase with random hexamers from Applied Biosystems (Foster City, CA). CT values were determined for the DIC, OGC, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using an Applied Biosystems Step-One Plus cycler and software. TaqMan® Gene Assay kits containing primer/probe sets for either the DIC (Slc25a10; GenBank Accession NM_133418.1; primer/probe set number Rn00591522), OGC (Slc25a11; GenBank Accession NM_022398.1; primer/probe set number Rn00574440), or GAPDH (endogenous control; GenBank Accession NM_017008.3; primer/probe set number Rn99999916) were purchased from Applied Biosystems. Optimum cDNA levels for all gene assays were determined to fall between 30 and 300 ng per PCR reaction. Final CT values were calculated for the DIC and OGC after normalization to

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Table 1 Basic parameters of renal function and health status of control and diabetic rats. Male Sprague–Dawley rats received an ip injection of streptozotocin (STZ; 60 mg/kg) and were maintained in metabolic cages with free access to food and water for up to 3 months. STZ rats developed polyuria 24 to 48 h after injection. A blood glucose (BG) level >250 mg/dl was considered indicative of the diabetic state. Rats with blood glucose >550 mg/dl were treated with insulin (1–2 unit IH daily) to maintain them as ketoacidosis free. Starting body weight (BW) was 208 ± 22 g and 219 ± 10 g for control and diabetic rats, respectively (mean ± SEM, n = 20 for each group). *Statistically significant difference (P b 0.05) from the corresponding value in control rats. Abbreviations: BW, body weight; KW, kidney weight; BG, blood glucose; BUN, blood urea nitrogen; NAG, N-acetyl-β-D-glucosaminidase; U-alb, urinary albumin; U-prot, urinary total protein. Parameter

Control

Diabetic

Control

1 month BW (g) KW (g) BG (mg/dl) BUN (mg/dl) NAG (mU/24 h) U-alb (mg/dl) U-prot (mg/24 h)

363 ± 22 (20) 2.47 ± 0.24 (20) 116 ± 17 (20) 18.9 ± 2.0 (8) 62.4 ± 11.0 (3) 7.25 ± 0.78 (9) 30.3 ± 4.6 (3)

Diabetic 3 months

280 ± 38* (20) 3.35 ± 0.65* (20) 574 ± 62* (20) 19.1 ± 2.6 (8) 576 ± 36* (3) 32.2 ± 9.4* (10) 126 ± 22* (3)

GAPDH CT levels. Relative expression levels were then calculated using the ΔCT method. Western blotting and antibodies. Rabbit anti-rat polyclonal antibodies to the DIC and OGC were purchased from Abcam (Cambridge, MA). Rabbit polyclonal antibody to 4-hydroxy-2-nonenal (HNE) Michael adducts of proteins was purchased from Calbiochem (La Jolla, CA, U.S.A.). Protein (100 μg) was loaded in the wells of 12% SDS polyacrylamide gels. After electroblotting of protein onto nitrocellulose paper, blots were blocked for 1 h in 5% milk powder solution and incubated overnight with the primary antibody. Blots were washed 3 times with Tris-buffered saline containing Tween 20 (TTBS) and

459 ± 12 (6) 3.12 ± 0.05 (6) 116 ± 10 (6) 17.3 ± 1.1 (5) 63.1 ± 13.1 (4) 5.55 ± 1.49 (6) 11.7 ± 3.7 (3)

312 ± 7* (7) 3.80 ± 0.13* (7) 600 ± 1* (7) 17.1 ± 1.6 (5) 1255 ± 221* (5) 113 ± 24* (6) 121 ± 20* (4)

incubated for 1 h with a polyclonal anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). Blots were then washed 3–6 times in TTBS and exposed for visualization on autoradiography film (Denville Scientific, Metuchen, PA) using enhanced chemiluminescence reagents (Pierce, Rockford, IL). Band density was derived using GelEval 1.2.2 software for Mac OS X. Lipid peroxidation assay. Malondialdehyde (MDA) formation was measured in mitochondrial suspensions according to the method of Gérard-Monnier et al. (1998), which involves reaction under mild acidic conditions of 1-methyl-2-phenylindole as the colorometric

Fig. 2. Hemotoxylin and eosin (H&E) staining of sections of kidneys from 1-month and 3-month control and diabetic rats. Light microscopy of the kidney (hematoxylin and eosin [H&E] sections) are shown. Magnification= 200×. Arrow “a” in panel C (diabetic at 1 month) indicates the mild thickening of the glomerular basement membrane, glomerular hypertrophy, and mesangial expansion as compared with control at 1 month (arrow “a” in panel A). Arrows labeled “a” in panels B and D indicate the more extensive mesangial expansion in kidneys from 3-month diabetic rats whereas arrows labeled “b” indicate proximal tubular hypertrophy and contraction of the luminal space in kidneys of 3-month diabetic rats (panel D) as compared to those of 3-month control rats (panel B).

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reagent with MDA to form a stable chromophore whose absorbance is measured at 586 nm. 1,1,3,3-Tetramethoxypropane (0.5–20 μM) was used as a standard to generate MDA. Data analysis. All values are expressed as means ± SE of measurements from the indicated number of individual preparations. Fisher's protected least significance difference t-test was performed to determine significant differences between pairs with P values b 0.05 considered significant. Results Basic renal and physiological state of diabetic rats Basic parameters of health status and renal function were determined in 1- and 3-month diabetic rats and age-matched control rats to determine any overt effects at a time point prior to (i.e., 1 month) and after (i.e., 3 months) the onset of diabetic nephropathy (Table 1). While diabetic animals did gain body weight over the 3-month time course, overall weight gain in diabetic rats (1.42-fold) was markedly less than that in control rats (2.21-fold). In contrast, aggregate kidney weight, both in actual terms and as a fraction of body weight, was substantially higher in diabetic rats at both 1-month and 3-month time points, consistent with the renal hypertrophy that is known to occur in diabetes. As far as renal function is concerned, no differences were observed in BUN values at either the 1-month or 3-month time points. Surprisingly, however, urinary NAG, U-alb, and U-prot levels were markedly higher in diabetic rats than in corresponding control rats. BUN, like serum creatinine, is considered to be a fairly insensitive indicator or

biomarker of renal injury that only increases when renal function is significantly compromised (Hoffmann et al., 2011). The increases in these other parameters due to diabetes were larger in 3-month diabetic rats as expected, consistent with the onset of frank nephropathy at 45 to 60 days post-administration of STZ. Tissue morphology was also assessed by H&E (Fig. 2) and PAS staining (Fig. 3). In both cases, even at the 1-month time point, changes in cellular and tubular structure were clearly evident. These changes include thickening of the mesangium and glomerular basement membrane, increased vacuole formation in proximal tubules, and contraction of epithelial luminal space. As with the biochemical markers that were positive, the morphological changes increased in severity with the progression to diabetic nephropathy, as shown by comparing changes in the 1-month and 3-month diabetic rats. These histology measurements confirm that our animal model develops the well-characterized changes in renal morphology reported by others (Kakkar et al., 1997; Makino et al., 2003). Mitochondrial function and GSH-dependent and cellular energetics enzyme activities in diabetic rat kidneys Respiratory function was assessed with succinate as substrate in isolated mitochondria from renal cortex of diabetic and control rats (Fig. 4). Our previous study (Zhong and Lash, 2007) showed that only oxygen consumption coupled to site II (i.e., with succinate as substrate) was affected by diabetes whereas that coupled to substrates for site I (i.e., the NADH dehydrogenase) or site III (i.e., the cytochrome oxidase) was unaffected. Some mitochondrial samples were also incubated with either tert-butyl hydroperoxide (tBH) or

Fig. 3. p-Amino Shiff base (PAS) staining of sections of kidneys from 1-month and 3-month control and diabetic rats. Light microscopy of kidney sections stained with periodic acid Schiff's base (PAS) and counterstained with hematoxylin are shown. Magnification = 200×. Mesangial expansion and glomerular hypertrophy are evident in kidneys of diabetic rats at 1 month (arrow a in panel C); these effects worsen in kidneys of diabetic rats at 3 months (compare arrow “a” in panels B and D). In kidneys from 3-month diabetic rats, one also observes a larger number of hypertrophied proximal tubules with contracted lumen (compare arrow “b” in panels B and D).

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Fig. 4. Effects of the diabetic state on respiratory function in renal mitochondria from 1-month and 3-month control and diabetic rats. Rates of oxygen consumption were measured in suspensions of mitochondria from renal cortex from 1-month and 3-month Control (Con) and STZ-induced diabetic (Dia) rats. S3 = State 3; S4 = State 4; RCR = Respiratory Control Ratio = S3/S4. Rates of oxygen consumption at coupling site II were measured with 3.3 mM succinate (in the presence of 5 μM rotenone) as substrate. Mitochondria were incubated for 15 min with either buffer (= Control), 0.2 mM tBH, or 0.2 mM MVK. Results are means ± SEM of measurements from 4 mitochondrial preparations from each group of rats. *Significant difference (P b 0.05) between corresponding samples from Con and Dia rats. †Significant difference (P b 0.05) between corresponding control and tBH- or MVK-treated mitochondria.

methyl vinyl ketone (MVK) to test whether or not either of two well characterized oxidants or mitochondrial toxicants differentially affected respiration in mitochondria from control and diabetic rats. As we found previously (Zhong and Lash, 2007), renal cortical mitochondria from 1-month diabetic rats exhibited significantly higher rates of State 3 respiration with succinate as substrate than those from control rats. Additionally, no difference was observed in rates of State 4 respiration, indicating that renal mitochondria from diabetic rats exhibited significantly higher RCR values as well. Respiration rates in renal mitochondria from 3-month diabetic and age-matched control rats were generally similar to those observed for the 1-month samples, except that the increase in State 3 respiration, although still statistically significant, was such that RCR values did not differ in mitochondria from 3-month control and diabetic rats. Both tBH and MVK caused similar extents of inhibition of State 3 respiration when comparing mitochondria from diabetic and control rats at the same time point. Renal mitochondria from 3-month diabetic and control rats, however, were notably less sensitive to inhibition

by these toxicants than those from 1-month diabetic and control rats, suggesting some degree of adaptation had occurred. Activities of a battery of GSH-related and mitochondrial enzymes were measured in mitochondria or the appropriate subcellular fraction from kidneys of 1-month and 3-month diabetic and control rats (Table 2). Although activities of GRD did not differ between diabetic and control mitochondria at either time point, GPX activity was significantly higher in renal mitochondria from diabetic rats at both time points. This difference is consistent with a higher level of oxidative stress without any improvement in antioxidant capacity. While activity of one enzyme of the citric acid cycle, MDH, was significantly elevated at 1-month in renal mitochondria of diabetic rats, that of another, SDH, was slightly but significantly decreased. In renal mitochondria of 3-month diabetic rats, however, there were no differences between diabetic and control rats in activities of either enzyme. Again, this disappearance of a difference at the 3-month time point suggests that some adaptation to the diabetic nephropathy state has occurred. As shown previously (Zhong and Lash, 2007), both mitochondrial and homogenate (primarily cytoplasm) GSH concentrations were significantly higher in 1-month diabetic rat kidneys (Fig. 5). While GSH

Table 2 Activities of GSH-dependent and cellular energetics-related enzymes in kidneys of 1-month and 3-month control and diabetic rats. Male Sprague–Dawley rats received an ip injection of streptozotocin (STZ; 60 mg/kg) and were maintained in metabolic cages with free access to food and water for 1 or 3 months. Mitochondrial fractions, when used as enzyme source, were isolated by standard differential centrifugation of renal cortical homogenates. Values are mU/mg protein and are means ±SEM of measurements from 3 control and 3 diabetic rats. *Statistically significant difference (P b 0.05) from the corresponding value in control rats. Abbreviations: GDH, glutamate dehydrogenase; GGT, γ-glutamyltransferase; GPX, glutathione peroxidase; GRD, glutathione disulfide reductase; GST, glutathione S-transferase; MDH, malic dehydrogenase; SDH; succinate:cytochrome c oxidoreductase. Enzyme

Source

Control

Diabetic

1 month GRD GPX GST GCS GGT MDH GDH SDH

Mitochondria Mitochondria Homogenate Cytoplasm Homogenate Mitochondria Mitochondria Mitochondria

99.4 ± 7.2 163 ± 26 149 ± 34 25.2 ± 2.8 3230 ± 290 367 ± 52 117 ± 15 99.2 ± 12.9

Control

Diabetic

3 months 97.8 ± 13.2 294 ± 34* 47.7 ± 8.9* 21.2 ± 2.8 2050 ± 500* 583 ± 102* 138 ± 14 72.3 ± 10.7*

92.0 ± 1.5 120 ± 14 104 ± 14 46.5 ± 0.7 2265 ± 465 575 ± 195 97.0 ± 22.4 103 ± 22

109 ± 13 235 ± 23* 112 ± 11 62.5 ± 6.6* 2290 ± 535 480 ± 25 92.8 ± 17.0 95.6 ± 9.0

Fig. 5. Concentrations of GSH in renal cortical homogenates and mitochondria of 1-month and 3-month control and diabetic rats. GSH contents in renal cortical homogenates and mitochondria were measured with the GSH-Glo™ kit from Promega (Madison, WI) and were quantified by chemiluminescence in a SpectraMax 2 plate reader using GSH as a standard. Results are means ± SEM of measurements from samples from 4 of each group of rats. *Significantly different (P b 0.05) from the corresponding sample from control rats.

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Table 3 Kinetic parameters for mitochondrial transport of GSH and dicarboxylates in kidneys of 1-month and 3-month control and diabetic rats. Male Sprague–Dawley rats received an ip injection of streptozotocin (STZ; 60 mg/kg) and were maintained in metabolic cages with free access to food and water for 1 month or 3 months. Mitochondria were isolated by differential centrifugation and used for measurement of transport of GSH, malonate, and 2-oxoglutarate (2-OG) using radiolabeled substrates. Values for Km are in mM and for Vmax are in nmol/min per mg protein. Results are derived from Eadie-Hofstee or Line-Weaver Burke plots and are means ± SEM of measurements from renal mitochondria from 4 control and 4 diabetic rats. *Significantly different (P b 0.05) from the corresponding value in renal mitochondria of control rats. Control Parameter Malonate Km Vmax 2-OG Km Vmax GSH Km Vmax

1 month

Diabetic 3 months

1 month

3 months

1.17 ± 0.10 0.73 ± 0.21

1.33 ± 0.15 0.68 ± 0.08

1.20 ± 0.08 0.56 ± 0.11

2.06 ± 0.22* 1.76 ± 0.12*

0.30 ± 0.05 0.096 ± 0.010

0.81 ± 0.06 0.097 ± 0.005

0.27 ± 0.04 0.16 ± 0.02*

2.70 ± 0.33* 0.35 ± 0.02*

0.48 ± 0.04 2.37 ± 0.14

0.71 ± 0.05 2.43 ± 0.20

0.41 ± 0.06 2.75 ± 0.31*

0.36 ± 0.05* 3.10 ± 0.44*

concentrations in both renal mitochondria and homogenates were still significantly higher in 3-month diabetics than corresponding controls, the extent of elevation was much less (58% vs. 33% elevation in diabetic renal mitochondria at 1-month and 3-month, respectively). Effects of diabetic nephropathy on mitochondrial GSH transport Previous preliminary studies suggested that the higher levels of mitochondrial GSH in kidneys from diabetic rats were in part attributed to higher rates of transport of GSH from the cytoplasm into the mitochondria. However, the kinetics of transport were not analyzed in detail and were only assessed at the 1-month time point. Because transport of GSH across the renal mitochondrial inner membrane is determined by at least two carriers (Chen and Lash, 1998; Chen et al., 2000; McKernan et al., 1991), effects of diabetes may be complex and not simply a combination of effects on each individual carrier. Accordingly, kinetic parameters for transport of malonate (DIC substrate), 2-OG (OGC substrate), and GSH were determined in renal mitochondria from 1month and 3-month diabetic and age-matched control rats (Table 3). Malonate transport exhibited no significant changes in kinetic parameters at 1 month but showed a 2.6-fold increase in Vmax and a 55% increase in Km in renal mitochondria of 3-month diabetic rats. 2-OG transport was more variable, showing a 66% increase in Vmax but no significant change in Km at 1 month but a 3.6-fold increase

in Vmax and a 3.3-fold increase in Km at 3 months. Effects on kinetic parameters of GSH transport were somewhat more complex, in that at the 1-month time point, there was no significant difference in Km but renal mitochondria from diabetic rats exhibited a modest, but statistically significant 16% increase in Vmax as compared to those from control rats. At the 3-month time point, the Km for GSH transport was actually nearly 50% lower and the Vmax 28% higher in renal mitochondria from diabetic rats as compared to those from control rats, equating to a 2.5-fold increase in catalytic efficiency (i.e., Vmax/Km value). To determine if the changes in mitochondrial GSH transport activity and kinetics in diabetic kidneys are due to changes in transporter gene expression, real-time quantitative PCR was used to measure expression of DIC and OGC mRNA (Table 4). Surprisingly, expression of both DIC and OGC mRNA was markedly higher in kidneys from 1-month control rats relative to those from diabetic rats (7.4- and 71-fold higher in kidneys from control rats, respectively). At the 3-month time point, in contrast, mRNA expression in kidneys of diabetic rats of the DIC was ~40% higher and that of the OGC was not significantly different than expression in kidneys from control rats. Because patterns of gene expression often do not correlate with activity or protein expression, expression of DIC and OGC protein was determined by Western blot analysis using specific antibodies to the carrier proteins (Fig. 6). Expression of DIC protein was 2.6fold higher in kidneys from 1-month diabetic rats as compared to control whereas that of OGC protein did not differ in kidneys of diabetic and control rats. In kidneys from 3-month diabetic rats, expression of DIC protein was 2-fold higher than that of control kidneys and that of the OGC still did not differ between the two groups. Expression of VDAC was used as a mitochondrial housekeeping protein to normalize results for carrier expression. Other parameters of oxidative defense and mitochondrial injury Besides the GSH redox system, two other proteins are important in mitochondrial redox homeostasis, SOD2 and Trx2. Expression of SOD2 protein did not differ in renal mitochondria from diabetic and control rats at 1-month but was 3.9-fold higher in renal mitochondria from diabetic rats at the 3-month time point. In contrast, protein expression of Trx2 was significantly but only slightly lower in renal mitochondria from diabetic rats at 1 month and did not differ between diabetic and control rats at 3 months (Fig. 7). Modification of proteins by various ROS can generate products that are detectable with specific antibodies to the modified amino acid residues. One such modification is formation of HNE adducts. Extracts of renal mitochondrial proteins from 1-month and 3-month diabetic and age-matched control rats were subjected to Western blot analysis using an antibody that detects HNE-adducted proteins

Table 4 Real-time quantitative PCR analysis of DIC and OGC mRNA expression in renal cortex of 1-month and 3-month control and diabetic rats. Male Sprague–Dawley rats received an ip injection of streptozotocin (STZ; 60 mg/kg) and were maintained in metabolic cages with free access to food and water for 1 month or 3 months. Primers were designed with the aid of Oligo 6.76 and the cDNA sequences published in GenBank™. Primer and labeled probe sets were from Applied Biosystems. An optimum cDNA concentration of 30 to 300 ng DNA/well was determined for both DIC and OGC. CT values are based on measurements from the indicated number of total RNA samples from kidney cortex of 1-month and 3-month control and diabetic rats and are means ± SEM. GAPDH CT values were used for correction: For 1-month samples, GAPDH CT values = 28.39 ± 1.53 (n = 9) for control rats and 28.99 ± 0.61 (n = 9) for diabetic rats; for 3-month samples, GAPDH CT values = 23.83 ± 0.14 (n = 12) for control rats and 25.31 ± 0.19 (n = 12) for diabetic rats. Relative gene expression values were calculated by the formula: 2−ΔCT (Diabetic) / 2−ΔCT (Control), where the ΔCT value is the difference between the CT value for the gene of interest and that of GAPDH. *Significantly different (P b 0.05) from corresponding values from control rat kidneys. Gene

Control

Diabetic

CT

DIC — 1 month DIC — 3 months OGC — 1 month OGC — 3 months

32.48 ± 0.29 27.96 ± 0.13 30.91 ± 0.30 28.61 ± 0.14

(9) (9) (9) (8)

ΔCT

CT

4.09 ± 0.36 4.13 ± 0.03 2.52 ± 0.12 4.78 ± 0.14

35.96 ± 0.16 28.93 ± 0.17 37.64 ± 0.63 29.97 ± 0.11

(9) (12) (9) (12)

ΔCT

Relative expression (Diabetic/ Control)

6.97 ± 0.18* 3.63 ± 0.03* 8.65 ± 0.21* 4.66 ± 0.04

0.136 1.415 0.014 1.088

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Fig. 6. Western blot analyses of DIC and OGC protein expression in renal cortical mitochondria of 1-month and 3-month control and diabetic rats. Renal cortical mitochondria were isolated from homogenates by differential centrifugation. Mitochondrial protein (100 μg) was applied to SDS gels and then underwent immunoblot analysis using commercially available polyclonal antibodies for the DIC and OGC. Expression of VDAC was measured as a control for normalization of expression. Results are from 3 separate 1-month and 3-month control and diabetic rats. Band density values were derived using GelEval 1.2.2 software for Mac OS X and are means ± SEM of 3 measurements for each set of control or diabetic samples.

(Fig. 8). Four major protein bands, at 70, 50, 42, and 35 kDa, were detected in all samples. Analysis of each band density showed markedly different patterns when comparing samples from diabetic and control rats at the two time points. In samples from 1-month diabetic and control rats, three of the four bands were significantly higher in diabetic rats than in controls and one band was significantly lower. In contrast, two bands from the 3-month diabetic rats were significantly lower and two did not differ as compared to controls. Samples were also analyzed with an antibody to nitrotyrosine residues on proteins, but no differences between the mitochondrial proteome from

control or diabetic rats were detected at either time point (data not shown), suggesting that reactive nitrogen species and so-called nitrosative stress do not play a role in the pathophysiology of diabetic nephropathy in this model under the conditions studied. As a final indicator of differences due to diabetes in redox status in renal mitochondria, baseline and tBH-induced MDA levels were measured (data not shown). No differences were observed in baseline MDA levels in diabetic and control renal mitochondria at either time point. Incubation of renal mitochondria with tBH produced the expected increases in MDA levels in all samples, but no significant

Fig. 7. Western blot analyses of SOD2 and Trx2 protein expression in renal cortical mitochondria of 1-month and 3-month control and diabetic rats. Renal cortical mitochondria were isolated from homogenates by differential centrifugation. Mitochondrial protein (100 μg) was applied to SDS gels and then underwent immunoblot analysis using commercially available polyclonal antibodies for SOD2 and Trx2. Expression of VDAC was measured as a control for normalization of expression. Results are from 3 separate 1-month and 3-month control and diabetic rats. Band density values were derived using GelEval 1.2.2 software for Mac OS X and are means ± SEM of 3 measurements for each set of control or diabetic samples.

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Fig. 8. Western blot analyses of HNE-adducted proteins in renal cortical mitochondria of 1-month and 3-month control and diabetic rats. Renal cortical mitochondria were isolated from homogenates by differential centrifugation. Mitochondrial protein (100 μg) was applied to SDS gels and then underwent immunoblot analysis using a commercially available polyclonal antibodies against HNE-Michael adducts of proteins. Results are from 3 separate 1-month and 3-month control and diabetic rats. Band density values were derived using GelEval 1.2.2 software for Mac OS X and are means ± SEM of 3 measurements for each set of control or diabetic samples.

differences in response were observed between diabetic and control rats at either 1 month or 3 months. Discussion The present study examined differences in redox status and mitochondrial function in renal mitochondria at two stages after the induction of diabetes, first at a time when chronic hyperglycemia is well established but nephropathy is not considered to exist (i.e., 1 month post-STZ injection), and then at a time when frank nephropathy does exist (i.e., 3-months post-STZ injection). A previous, preliminary study of ours (Zhong and Lash, 2007) showed that at 1 month post-STZ injection, mitochondrial respiration and GSH content were higher in kidneys of diabetic rats as compared to those of control rats. Here we extended that study and tested the hypothesis that as diabetic nephropathy progresses, adaptive changes will occur in mitochondrial energetics and thiol redox status but that these changes will be insufficient to prevent oxidative stress and ultimately decreased renal function. Although the STZ-treated rat is a well-established and accepted model of Type 1 diabetes and rats given STZ typically develop nephropathy within 45 to 60 days of becoming hyperglycemic (Tesch and Allen, 2007), little information was available about basic renal function in hyperglycemic, pre-nephropathy rats or as diabetic animals progress to nephropathy. Accordingly, the present study evaluated basic parameters of renal and specifically PT function at 1-month and 3-months post-STZ and in age-matched control rats. Perhaps the most surprising result was that three markers of renal and PT function, urinary NAG, U-alb, and U-prot, were significantly and markedly higher in 1-month diabetic rats than in age-matched control rats. Moreover, differences in these parameters between diabetic and control rats were even greater at the 3-month time point, consistent with the progression to nephropathy. These data were supported by microscopy, which showed morphological changes consistent with diabetic

nephropathy even after 1 month of hyperglycemia. Kakkar et al. (1997) showed mesangial expansion beginning around 6 weeks after induction of diabetes with STZ and Tone et al. (2005) reported albuminuria at 4 weeks post-STZ injection. Hence, it is clear that pathophysiological changes begin very early after the onset of hyperglycemia in the STZ rat model. Despite the progression to nephropathy between the 1-month and 3-month time points, other parameters that were initially different from those in control rats at 1 month either exhibited smaller differences from controls or a complete normalization at 3 months. This response suggests that there is some degree of adaptation to the diabetic state that occurs with time. For example, while the rate of State 3 oxygen consumption was significantly higher in renal mitochondria from diabetic rats at both the 1-month and 3-month time points, the RCR value was only significantly higher in renal mitochondria at 1 month. This partial normalization of respiratory rate is reflected in activities of three citric acid cycle enzymes (cf. Table 2), which were markedly higher in diabetic than control mitochondria at 1 month but not at 3 months. Thus, while there is still some increased flux of electrons through the respiratory chain in 3-month diabetic renal mitochondria, many of the processes dependent on the respiratory chain have been largely normalized. The chronic hyperglycemia associated with diabetes has been clearly associated with increased levels of ROS, oxidative stress, and changes in mitochondrial function (Brownlee, 2001; Hakim and Pflueger, 2010; Ibrahim and Hostetter, 1997; Obrosova et al., 2003; Park et al., 1997; Prabhakar et al., 2007; Rolo and Palmeira, 2006). Many of the changes that occur in redox status in the diabetic kidney can be viewed as compensatory. GSH, being the predominant intracellular non-protein thiol compound, and many of its associated enzymes, were demonstrated to change in the diabetic state. Of particular importance to how the diabetic kidney responds to oxidative stress and exposures to potentially nephrotoxic drugs and chemicals, is how GSH status changes in mitochondria. Although one may

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have predicted a depletion or oxidation of the GSH pool, the opposite was found. Renal cellular concentrations of GSH, and particularly those in mitochondria, were significantly higher in both 1-month and 3-month diabetic rats as compared to the corresponding control rats. GSH concentrations are controlled by activity of two processes, GCS and membrane transporters. GCS in the renal proximal tubule is localized predominantly, if not exclusively, in the cytoplasm (McKernan et al., 1991). Its activity is regulated primarily by feedback inhibition by GSH (Anderson, 1998) and its expression is regulated by the antioxidant response element (Mulcahy et al., 1997; Toroser et al., 2006). Although total cellular and mitochondrial GSH concentrations were elevated in kidneys from 1-month diabetic rats, GCS activity did not differ in renal cortical cytoplasm from diabetic or control rats. At 3 months, however, GCS activity was 34% higher in renal cortical cytoplasm from diabetic rats, consistent with a long-term adaptive response. Because the mitochondrial GSH pool is derived from the cytoplasm by function of two mitochondrial inner membrane transporters (Lash, 2006), changes in GCS activity can translate into changes in mitochondrial GSH concentrations by causing changes in substrate supply for the transporters. However, the pattern of change in GCS activity did not match that of GSH, suggesting that GSH synthesis was not a major factor in the elevated concentrations of mitochondrial GSH in diabetes. Hence, the focus to explain the mechanism must be on the two inner membrane carriers, the DIC and OGC, which transport GSH from the cytoplasm into the mitochondrial matrix. The two mitochondrial GSH transporters were compared in renal mitochondria of 1-month and 3-month diabetic and age-matched control rats at three levels: Activity, gene expression, and protein expression. Two considerations make regulation of mitochondrial GSH transport complex. First, transport occurs by the combined function of two carriers (Lash, 2006). Second, transport of GSH is strongly influenced by that of various dicarboxylates and other citric acid cycle intermediates. Regarding the first consideration, the complexity lies in the potential for changes in one of the carriers to be compensated by opposing changes in the other carrier or simply difficulty in linking functional change with effects on one carrier. Regarding the second consideration, this principle was originally enunciated by us in a recent study (Lash et al., 2007) that identified the organic anion transporter 3 (Oat3; Slc22a8) as being one of the basolateral plasma membrane carriers that mediate uptake of GSH from the renal plasma and interstitial space into the PT cell. The concept, recently updated (Lash, 2011), is that levels of dicarboxylates, in particular 2-OG, and metabolic fluxes through the citric acid cycle and mitochondrial electron transport chain influence rates of mitochondrial GSH transport because 2-OG and other dicarboxylates are directly coupled to several of the carriers for GSH transport. Thus, at the basolateral plasma membrane, Oat3 functions to exchange intracellular 2-OG for extracellular GSH. At the mitochondrial inner membrane, the OGC functions to exchange mitochondrial 2-OG with other dicarboxylates or GSH from the cytoplasm, and the DIC functions to exchange mitochondrial inorganic phosphate with various dicarboxylates or GSH from the cytoplasm. Changes in rates of intermediary metabolism would influence dicarboxylate levels, thereby changing availability of co-substrates for the above-mentioned carriers. At the level of activity, we found that rates of GSH uptake across the mitochondrial inner membrane were modestly elevated at both 1 month and 3 months post-STZ as compared to their respective controls. By measurement of transport kinetics for malonate and 2-OG, effects on the DIC and OGC, respectively, could be separately evaluated. The results (cf. Table 3) showed that changes in activity of both carriers could contribute to the changes in GSH uptake, although the relative role of each carrier differed substantially at the two time points. Specifically, in renal mitochondria from 1-month diabetic and control rats, there was no significant difference in DIC activity but OGC activity was 1.6-fold higher in diabetic samples; in renal

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mitochondria from 3-month diabetic and control rats, in contrast, activities of both the DIC and OGC were >2-fold higher in diabetic samples as compared to control samples. Surprisingly, Km values for malonate and 2-OG uptake were markedly higher in renal mitochondria from 3-month diabetic rats whereas the Km for GSH uptake was actually lower by almost 50%. Inasmuch as we previously showed that the DIC and OGC account for > 80% of mitochondrial GSH transport (Chen and Lash, 1998; Chen et al., 2000), the differences in effects on transporter function with different substrates were unexpected but not unprecedented, as enzymes may behave differently under certain circumstances with different classes of substrates. Assessment of gene and protein expression and comparison with transporter kinetic data further highlights the complexity of determining factors that regulate mitochondrial GSH transport. Real-time quantitative PCR analysis of mRNA levels showed that gene expression for both carriers was significantly lower (not higher) in kidney samples from 1-month diabetic rats as compared to controls. In contrast, the level of mRNA for the DIC was modestly higher in kidneys of 3-month diabetic rats as compared to those in control rats whereas OGC mRNA did not significantly differ between kidneys of control and diabetic rats. Western blot analysis of protein expression showed substantially higher levels of DIC protein in kidneys of diabetic rats at both time points but no differences in OGC protein levels. While it is not necessarily uncommon that patterns of mRNA levels do not correspond with those of protein expression or activity, the results were surprising. Potential explanations for higher protein levels but lower mRNA levels may include increased translation efficiency and/or post-translational effects such as inhibited protein degradation. Taken together, the transporter activity and expression data combined with consideration of differences in mitochondrial metabolism suggest that while some of the increase in mitochondrial GSH content associated with diabetes is due to direct changes in expression of the transporters, most of the increase in mitochondrial GSH content is due to the hypermetabolic state of the diabetic renal proximal tubule, which results in greater delivery of dicarboxylate co-substrate to the GSH carriers, thereby increasing their turnover. A final issue that was examined concerned the relative level of oxidative stress in diabetic as compared to control kidney mitochondria. Our earlier study in isolated PT cells (Zhong et al., 2009) provided strong evidence for increased oxidative stress due to diabetes. In the present study, two types of assays were conducted in isolated renal mitochondria to address this issue, assessment of oxidatively modified proteins and measurement of lipid peroxidation. Two types of modified proteins were measured, HNE- and 3-nitrotyrosine-adducted proteins. No differences in 3-nitrotyrosine-adducted proteins were detected, suggesting a lack of involvement of reactive nitrogen species in the pathology of diabetic nephropathy in this particular model. In contrast, significant differences in quantities and patterns of HNE-adducted mitochondrial proteins were evident, suggesting that some redox changes exist in diabetic kidney and that ROS are linked to some of the key biochemical changes that occur. Measurement of MDA as a marker for lipid peroxidation, however, showed no differences between either basal or tBH-induced lipid peroxidation in renal mitochondria of diabetic or control kidneys at either time point. Increased GCS activity at 3 months was also interpreted as being consistent with long-term adaptation to changes in redox status. In conclusion, the present study has demonstrated significant differences in mitochondrial function and redox status between kidneys of control and diabetic rats. Significant signs of renal dysfunction were detected in 1-month diabetic rats, indicating that even prior to development of true nephropathy there are functional changes resulting from chronic hyperglycemia. Some of these functional changes predictably worsened as animals progressed to true diabetic nephropathy. However, several other biochemical parameters that exhibited significant changes at 1 month, including rates of mitochondrial respiration and citric acid cycle enzymes and changes in GSH redox status, showed adaptive

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responses at 3 months. Change in mitochondrial GSH transport, which results in higher content of GSH in renal mitochondria from diabetic rats as compared to those from control rats, is due to a complex interplay of transcriptional and translational control and mass action effects due to significant changes in dicarboxylate supplies. Although not all assays of redox status clearly show enhanced oxidative stress, some indicators such as HNE-protein adduct levels, elevated GCS and GPX activities without concomitant increases in GRD activity, and impaired oxidative phosphorylation (relative decrease in RCR at 3 months), are consistent with redox status and GSH in particular being an appropriate therapeutic target for future studies to improve the metabolic phenotype of the diabetic kidney. Conflict of interest None. Acknowledgments This research was supported by Department of Defense grant PR064340 (Contract Number W81XWH-07-1-0453). Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.taap.2011.10.021. References Alderson, N.L., Chachich, M.E., Frizzell, N., Canning, P., Metz, T.O., Januszewski, A.S., Youssef, N.N., Stitt, A.W., Baynes, J.W., Thorpe, S.R., 2004. Effect of antioxidants and ACE inhibition on chemical modification of proteins and progression of nephropathy in the streptozotocin diabetic rat. Diabetologia 47, 1385–1395. Anderson, M.E., 1998. Glutathione: an overview of biosynthesis and modulation. Chem. Biol. Interact. 111–112, 1–14. Beisswenger, P.J., Drummond, K.S., Nelson, R.G., Howell, S.K., Szwergold, B.S., Mauer, M., 2005. Susceptibility to diabetic nephropathy is related to dicarbonyl and oxidative stress. Diabetes 54, 3274–3281. Brownlee, M., 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414, 813–820. Chander, P.N., Gealekman, O., Brodsky, S.V., Elitok, S., Tojo, A., Crabtree, M., Gross, S.S., Goligorsky, M.S., 2004. Nephropathy in Zucker diabetic fat rat is associated with oxidative and nitrosative stress: prevention by chronic therapy with a peroxynitrite scavenger ebselen. J. Am. Soc. Nephrol. 15, 2391–2403. Chen, Z., Lash, L.H., 1998. Evidence for mitochondrial uptake of glutathione by dicarboxylate and 2-oxoglutarate carriers. J. Pharmacol. Exp. Ther. 285, 608–618. Chen, Z., Putt, D.A., Lash, L.H., 2000. Enrichment and functional reconstitution of glutathione transport activity from rabbit kidney mitochondria: further evidence for the role of the dicarboxylate and 2-oxoglutarate carriers in mitochondrial glutathione transport. Arch. Biochem. Biophys. 373, 193–202. Estabrook, R.W., 1967. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10, 41–47. Fioretto, P., Bruseghin, M., Berto, I., Gallina, P., Manzato, E., Mussap, M., 2006. Renal protection in diabetes: role of glycemic control. J. Am. Soc. Nephrol. 17, S86–S89. Fleischer, S., Fleischer, B., 1967. Removal and binding of polar lipids in mitochondria and other membrane systems. Methods Enzymol. 10, 406–433. Geiss, L.S., Herman, W.H., Goldschmid, M.G., DeStefano, F., Eberhardt, M.S., Ford, E.S., German, R.R., Newman, J.M., Olson, D.R., Sepe, S.J., 1993. Surveillance for diabetes mellitus: United States, 1980–1989. MMWR CDC Surveill. Summ. 42, 1–20. Gérard-Monnier, D., Erdelmeier, I., Régnard, K., Moze-Henry, N., Yadan, J.C., Chaudière, J., 1998. Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation. Chem. Res. Toxicol. 11, 1176–1183. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Hakim, F.A., Pflueger, A., 2010. Role of oxidative stress in diabetic kidney disease. Med. Sci. Monit. 16, RA37–RA48. Halestrap, A.P., 1975. The mitochondrial pyruvate carrier: kinetics and specificity for substrates and inhibitors. Biochem. J. 148, 85–96.

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