Kidney function declines with advancing age and mitochondria have been implicated. In the present study we have examined the integrated function of mitochondria isolated from kidneys of 6- and 24-month-old Fischer 344 rats. OXPHOS (oxidative phosphorylation) of intact mitochondria and cytochrome c oxidase activity in permeabilized mitochondria were determined with polarographic assays. The activities of the ETC (electron transport chain) complexes and the cytochrome content in solubilized mitochondria were measured using spectrophotometric methods. The respiratory complexes were evaluated with blue native gel electrophoresis. Mitochondrial preparations were evaluated by immunoblotting for cytochrome c, Smac/Diablo and VDAC (voltage-dependent anion channel). Mitochondrial morphology was examined by electron microscopy. OXPHOS of mitochondria isolated from 24-month-old animals was decreased 15–25% with complexes I, II, III and IV, and fatty acid substrates. The electron microscopic appearance of mitochondria, the activity of the ETC complexes and the protein abundance of individual complexes and supercomplexes were unchanged. The content of cytochrome c was decreased by 37% in aged mitochondria, as determined by spectrophotometric methods and confirmed with immunoblotting. Polarographic determination of cytochrome c oxidase activity with endogenous cytochrome c demonstrated a 23% reduction in aged mitochondria, which was corrected with the addition of exogenous cytochrome c. Renal mitochondrial OXPHOS decreased with aging in the Fischer 344 rat. Decreased mitochondrial cytochrome c content is a major factor contributing to the OXPHOS defect of mitochondria isolated from kidneys of elderly animals.

INTRODUCTION

The age-related decline in kidney function in humans has been recognized for many decades [1,2], and the prevalence of reduced kidney function in individuals >64 years of age is between 23.4 and 35.8% [3]. The incidence of acute kidney injury is also increased in older individuals for a variety of reasons [4]. The kidney has high energetic demands largely related to its active solute transport functions, accounting for 10% of whole-body oxygen consumption, and as a whole the kidney utilizes mitochondrial OXPHOS (oxidative phosphorylation) to generate approx. 95% of its ATP [5]. Small amounts of ROS (reactive oxygen species) are produced in the mitochondria as a by-product of electron flux through the respiratory chain. The mitochondrial theory of aging states that mitochondrial ROS production leads to stochastic oxidative damage over time with the accumulation of mtDNA (mitochondrial DNA) mutations and decrements in mitochondrial function leading to the phenotypic changes of aging [6]. These features place the mitochondria in a central role for many of the mechanisms implicated in the progression of the aging process [6,7].

Rat models have provided valuable insights to the study of aging. Age-related decrements in mitochondrial OXPHOS and the ETC (electron transport chain) have been well described in several organs and tissues of the rat, but relatively little attention has been given to the kidney. One study used immunohistochemical methods to detect the age-related appearance of ETC-deficient renal tubular epithelial cells in the rat kidney [8]. The authors demonstrated that these effects were due to mtDNA deletions in the ETC-deficient cells, presumably generated via ROS-mediated damage. There is evidence to support an age-related increase in ROS production within the rat kidney, and a decline in the enzymes of the antioxidant systems [9,10]. However, to date there has been no detailed biochemical examination of the effects of aging on renal mitochondrial OXPHOS and ETC.

Therefore we have undertaken the present studies to examine isolated renal mitochondria for age-related decrements in function, as have been described in other organs and tissues. In the present study we utilize spectrophotometric assays to evaluate the enzyme activities of the individual components of the ETC and mitochondrial abundance of cytochromes. Blue native gel electrophoresis is used to determine the relative abundance of the individual respiratory complexes, as well as supercomplexes in mitochondria isolated from 6- and 24-month-old animals. Furthermore, polarographic assays of oxygen consumption using freshly isolated renal mitochondria permits the evaluation of efficiency of ATP production in intact mitochondria. These assays depend upon the function of substrate transporters, dehydrogenases and respiratory complexes, while providing measures of the integrity of the isolated mitochondria via the RCR (respiratory control ratio) and coupling to the phosphorylation apparatus via ADP/O ratios (number of ADP molecules added for each oxygen atom consumed). The use of a panel of metabolic substrates in conjunction with inhibitors and uncouplers of the respiratory chain permit the biochemical localization of the site of defects in mitochondrial OXPHOS.

MATERIALS AND METHODS

Reagents

All chemicals were purchased from Sigma–Aldrich, except where otherwise noted, and were of the highest purity available.

Animals

Male Fischer 344 rats were obtained from the National Institute of Aging colony (Harlan Laboratories) under an approved IACUC (Institutional Animal Care and Use Committee) protocol (2006-0080). Rats were housed in the animal facility at the VA Medical Center and the investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The rats were fed ad libitum a 70717 NIH-31 Open Formula Rat diet (18% protein, 4% fat, 5% fibre and 8% ash) and had free access to water. Animals were obtained at the age required for the study and were used after allowing a 7 day adjustment period.

Mitochondrial isolation

Rats were weighed and then killed at 6 or 24 months by decapitation according to the approved IUPAC protocol (2006–0080). Mitochondrial isolation was adapted from previously reported methods [11], and is briefly summarized as follows. Kidneys were extirpated and rinsed in ice-cold MSM [220 mM D-mannitol, 70 mM sucrose and 5 mM Mops (pH 7.4)]. Kidneys were decapsulated, rinsed in MSM and weighed. Kidneys were minced, rinsed twice in MSM and suspended in 10 ml of MSM+ (MSM plus 2 mM EDTA and 0.2% defatted BSA) per g of wet weight. The suspension was homogenized with three strokes of a loose-fitting pestle in a Potter–Elvehjem homogenizer at ~1000 rev./min. The homogenate was centrifuged at 300 g in a Sorvall SS-34 rotor at 4 °C, and supernatant was decanted through gauze. The low-speed pellet was resuspended in MSM+, homogenized and centrifuged, as above. The supernatant was decanted through gauze and combined with the first supernatant. Combined supernatants were centrifuged at 7000 g using the SS-34 rotor at 4 °C. Supernatant was decanted and the pellet was resuspended in MSM and centrifuged at 7000 g two more times. The final mitochondrial pellet was resuspended in 0.2 ml of MSM/g of wet weight of starting material. The protein concentration was determined using biuret reagent with BSA as a standard. Mitochondria were maintained on ice until used in subsequent assays on preparations performed that day, or stored at −80 °C for future use.

OXPHOS assays

Measurements of OXPHOS were performed as previously described [12] on freshly isolated mitochondria in a Clark-type oxygen electrode (Strathkelvin). Assays were performed by adding mitochondria to 0.5 ml of air-saturated respiration buffer [100 mM KCl, 5 mM KH2PO4, 1 mM EGTA, 1 mg/ml defatted BSA and 50 mM Mops (pH 7.4)] equilibrated to 30 °C. Rotenone solubilized in DMSO was added in a volume of 5 μl to the respiration buffer prior to assays using the substrates, succinate, DHQ (duroquinol) and TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine), at a final concentration of 7.5 μM. Endogenous substrates were depleted by the addition of ADP to a concentration of 75 μM. Substrate or substrate combinations were then added to the chamber in volumes ranging from 5 to 25 μl per component to achieve the final concentrations as follows: glutamate (20 mM), malate (5 mM), pyruvate (10 mM), malonate (10 mM), α-oxoglutarate (10 mM), succinate (20 mM), DHQ (1 mM), TMPD (1 mM), ascorbate (10 mM), acetylcarnitine (5 mM), palmitoylcarnitine (0.04 mM), carnitine (5 mM) and palmitoyl-CoA (0.02 mM). State 3 (ADP-stimulated) and State 4 (ADP-limited) respiratory rates were recorded after the addition of ADP to a concentration of 150 μM or 75 μM for succinate and DHQ. State 3 rates were recorded in the presence of a high ADP concentration (2 mM). Rates of uncoupled respiration were recorded after the addition of dinitrophenol to a concentration of 0.2 mM (Fisher Scientific). RCRs (State 3 divided by State 4) reflects the integrity of the mitochondrial preparation and control of oxygen consumption by phosphorylation (‘coupling’). The ADP/O ratios is an index of the efficiency of OXPHOS [13,14].

Citrate synthase

Citrate synthase activity was determined as described previously [15], using a diode array spectrophotometer at a wavelength of 412 nm.

ETC assays

Samples of fresh RKM (rat kidney mitochondria) were treated with 10 mg of cholate/mg of mitochondrial protein, taken to a final concentration of 1 mg/ml in MSM-EDTA buffer supplemented with 1 μl/ml mammalian protease inhibitor cocktail. For the assay, the samples were diluted 1:10. For COX (cytochrome c oxidase) activity, 0.1 mg of fresh intact mitochondria were suspended in 100 mM KCl, 50 mM Mops and 0.5 mM EGTA (pH 7.4), and assayed with and without dodecyl β-D-maltoside. ETC enzyme activities were measured spectrophotometrically as specific donor–acceptor oxidoreductase activities in 0.1 M phosphate buffer. Both donor and acceptors span specific regions of the ETC [1618]. Rotenone-sensitive NADH-cytochrome c reductase assesses complexes I and III. NADH coenzyme Q reductase was measured as the rotenone-sensitive oxidation of NADH with decylubiquinone as the acceptor, and assesses only complex I. NADH ferricyanide reductase measures NADH dehydrogenase and mitochondrial outer membrane cytochrome b5 (rotenone-insensitive NADH-reductase). Antimycin A-sensitive succinate-cytochrome c reductase assesses complexes II and III. Complex II activity was measured as the TTFA (thenoyltrifluoroacetone)-sensitive reduction of 2,6-dichlorophenolindophenol with succinate as the substrate, whereas total complex II was measured in the same manner, but with duroquinone added as a source of exogenous coenzyme Q. Succinate dehydrogenase measures the first two subunits of complex II [19]. Decylubiquinol-cytochrome c oxidoreductase was measured as the antimycin A-sensitive reductase to assess complex III [20]. COX was measured as the oxidation of reduced cytochrome c and expressed as the first-order rate constant.

Electron microscopy

Electron microscopic analysis was performed as described in [17], with changes as noted below. RKM were prepared from 6- and 24-month-old animals as described above. An aliquot of RKM was added to an equal volume of phosphate-buffered, half-strength Karnovsky's fixative, mixed and immediately spun down for 30 s in a microcentrifuge. The fixation continued in fresh one-quarter-strength Karnovsky's fixative for a total of 2 h at room temperature (23 °C). The pellets were thoroughly rinsed in distilled water, then post-fixed for 2 h in an unbuffered 1:1 mixture of 2% osmium tetroxide and 3% potassium ferricyanide. After rinsing with distilled water, the specimens were soaked overnight in an acidified solution of 0.25% uranyl acetate. After another rinse in distilled water, they were dehydrated in ascending concentrations of ethanol, passed through propylene oxide and embedded in Poly/Bed 812 embedding media (Polysciences). Thin sections (70 nm) cut on a RMC MT6000-XL ultramicrotome were mounted on Gilder square 300 mesh nickel grids (Electron Microscopy Sciences) and then sequentially stained with acidified methanolic uranyl acetate followed by lead. These were coated in a Denton DV-401 carbon coater (Denton Vacuum LLC), and examined in a Zeiss CEM 902 electron microscope.

Measurement of cytochrome spectra

The mitochondrial content of cytochromes aa3, b, c1 and c was quantitatively determined using the difference spectra according to the method of Williams [21]. Briefly, mitochondria stored at −80 °C were thawed on ice and 0.25 mg of mitochondrial protein was solubilized in reaction buffer [2.1% sodium deoxycholate, 5 mM sodium L-ascorbate and 25 mM NaH2PO4 (pH 7.0)]. The absorption spectra between 500 and 660 λ were recorded using an HP 8453 UV–visible ChemStation spectrophotometer with ChemStation software for each sample when oxidized and reduced. The difference spectra (reduced−oxidized) were used for the solution of simultaneous equations to quantitatively determine the content of these cytochromes in the mitochondrial samples as described by Williams [21].

Complex IV oxidase assay

Mitochondria stored at −80 °C were thawed on ice and 0.25 mg of mitochondrial protein was diluted to a final concentration of 1 mg/ml in KPi/EDTA buffer [20 mM KH2PO4 and 0.1 mM EDTA (pH 7.4)]. KPi buffer without or with exogenous cytochrome c, at a final concentration of 32 μM (Calbiochem) was added to the chamber of a Clark-type oxygen electrode (Strathkelvin). After equilibration of 500 μl of KPi buffer in the reaction chamber to 30 °C, 25 μl of KPi-treated mitochondria were added and baseline rates recorded. Substrate was added (1 mM TMPD+10 mM ascorbate) and the substrate-stimulated rate was recorded. Sodium azide (at 2 mM) was added and the azide-insensitive rate was recorded. The complex IV azide-sensitive rates were determined for each assay by determining the difference between the substrate-stimulated rates and the azide-insensitive rates.

Blue native gel electrophoresis

Blue native gel electrophoresis was performed according to previously reported methods [22] and are briefly summarized below. An aliquot of freshly isolated mitochondria containing 300 μg of mitochondrial protein was resuspended in buffer and solubilized with digitonin (Calbiochem) or dodecyl maltoside by incubation for 30 min on ice. Unsolubilized membranes were pelleted by centrifugation for 10 min at 100000 rev./min in an Airfuge® ultracentrifuge. The supernatant was removed and 20 μl of sample buffer was added as described previously [22]. Samples were divided into 20 μl aliquots and stored at −80 °C for future use. Samples were loaded (75 μg in a volume of 15 μl) on to a 3–12% precast NativePAGE Novex Bis-Tris gradient gel (Invitrogen) for analysis of digitonin-solubilized samples or a 4–16% precast NativePAGE Novex Bis-Tris gradient gel (Invitrogen) for analysis of dodecyl-maltoside-solubilized samples. Samples were separated with electrophoresis at a voltage of 75 V for a total of 1300 Vh. Gels were destained and imaged with the GeneGenius Bioimaging System (Syngene). Gel images were processed with ImageJ software for background subtraction using a rolling ball algorithm based upon previous methods [23], with a rolling ball radius of 50 pixels. Background-subtracted images were imported into MultiGauge v2.0 software (Fujifilm) for calculation of band density by calculation of the area under the curve for each of the respective respiratory complex bands.

Immunoblotting

Mitochondrial samples, previously stored at −80 °C, were thawed on ice and solubilized in SDS/PAGE loading buffer and boiled for 5 min. Samples were loaded on to a 12.5% acrylamide gel (50 μg of mitochondrial protein/lane) and separated with SDS/PAGE. Separated proteins were transferred on to PVDF membrane and allowed to air-dry before blocking for 1 h at 21 °C in TBS [Tris-buffered saline (25 mM Tris base, 137 mM NaCl, 3 mM KCl, pH 7.4 with HCl)] with 5% non-fat dried skimmed milk. Membranes were then incubated for 2 h at 21 °C in blotting buffer [1% non-fat dried skimmed milk and 0.1% Tween 20 in TBS (pH 7.4)] with primary antibodies against cytochrome c at 1:500 (Epitomics), Smac/Diablo at 1:1000 (Epitomics) and VDAC (voltage-dependent anion channel) at 1:1000 (Mitosciences). Membranes were washed three times for 10 min at 21 °C in blotting buffer, incubated with HRP (horseradish peroxidase)-conjugated secondary antibodies at 1:5000 in blotting buffer for 1 h at 21 °C, followed by three washes in blotting buffer and one wash in TBS. A chemiluminescent detection kit (Santa Cruz Biotechnology) was used for HRP detection, and membranes were exposed to film for times appropriate to obtain non-saturating exposures and developed. Film was scanned as TIFF files and the band density was determined using ImageJ software available from the National Institutes of Health.

Statistics

Results are reported as means±S.D. Comparisons between groups are made by a two-tailed Student's t test and reported as a two-tailed P value. Statistical significance is considered to be P<0.05.

RESULTS

Comparisons were made between the 6- and 24-month-old animals for body weight, kidney weight, mitochondrial yield and the activity of the mitochondrial marker enzymes citrate synthase and succinate dehydrogenase in isolated mitochondria (Table 1). There was no significant difference in body weight between the two groups. The kidney size, as expressed in g per wet weight, was significantly greater in the 24-month-old animals. The mitochondrial yield, as expressed in mg of mitochondrial protein per gram of wet weight of kidney tissue, was significantly lower in the 24-month-old animals. The efficiency of mitochondrial recovery was 22±4% compared with 23±8% in the 6- and 24-month-old groups respectively. Recovery was determined by the percentage of citrate synthase activity recovered in the final mitochondrial isolate obtained per g of kidney weight to the total citrate synthase activity in the initial homogenate per g of kidney weight. The decreased mitochondrial yield per g of kidney in the context of equivalent recovery efficiency suggest that the lower yield is a reflection of decreased mitochondrial protein content per g of kidney tissue in the 24-month-old animals. The relative equivalence of the mitochondrial preparations, with respect to mitochondrial purity, was assessed by the activities of the mitochondrial marker enzymes citrate synthase and succinate dehydrogenase. There were no significant differences in the mitochondrial preparations obtained from the 6- and 24-month-old animals for either marker enzyme (Table 1). Citrate synthase activity per mg of wet weight of kidney and succinate dehydrogenase activity per mg of wet weight of kidney were lower in the older animals, but this trend did not reach statistical significance.

Table 1
Body and kidney weight, mitochondrial yield and mitochondrial marker enzymes in 6- and 24-month-old animals

Values are means±S.D.

Measurement 6-Month-old (n=8) 24-Month-old (n=8) P 
Body weight (g) 381.3±6.4 386.3±28.8 0.6386 
Kidney weight (g) 2.2±0.2 2.7±0.6 0.0340 
Citrate synthase (nmol/min per mg of wet weight) 23.8±3.2 20.2±5.6 0.1895 
Succinate dehydrogenase (nmol/min per mg of wet weight) 8.5±1.2 6.8±2.3 0.1317 
Mitochondrial yield (mg/g of wet weight) 16.8±1.0 12.3±2.3 0.0002 
Citrate synthase (nmol/min per mg of mitochondrial protein) 301.4±51.0 354.7±78.8 0.1306 
Succinate dehydrogenase (nmol/min per mg of mitochondrial protein) 139.8±38.1 120.8±26.6 0.2668 
Measurement 6-Month-old (n=8) 24-Month-old (n=8) P 
Body weight (g) 381.3±6.4 386.3±28.8 0.6386 
Kidney weight (g) 2.2±0.2 2.7±0.6 0.0340 
Citrate synthase (nmol/min per mg of wet weight) 23.8±3.2 20.2±5.6 0.1895 
Succinate dehydrogenase (nmol/min per mg of wet weight) 8.5±1.2 6.8±2.3 0.1317 
Mitochondrial yield (mg/g of wet weight) 16.8±1.0 12.3±2.3 0.0002 
Citrate synthase (nmol/min per mg of mitochondrial protein) 301.4±51.0 354.7±78.8 0.1306 
Succinate dehydrogenase (nmol/min per mg of mitochondrial protein) 139.8±38.1 120.8±26.6 0.2668 

OXPHOS was evaluated in freshly isolated intact mitochondria. The State 3 and State 4 respiratory rates, the RCR, the rate of respiration obtained in the presence of high ADP (2 mM), the uncoupled rate of respiration obtained in the presence of DNP and the ADP/O ratio were recorded, as shown for the substrate combination of glutamate and malate (Table 2). Using the substrate combination of glutamate and malate, the State 3, State 4 and high [ADP] respiratory rates were decreased in the 24-month-old animals by 13%, 21% and 13% respectively (Table 2). The uncoupled respiratory rate, the RCR, a metric of mitochondrial integrity and coupling, and the ADP/O ratio, a reflection the control and efficiency of the phosphorylation apparatus, were not significantly different between the two groups (Table 2).

Table 2
Polarographic assay of 6- and 24-month-old rat kidney mitochondria using the substrate combination of glutamate and malate

Values are means±S.D.

Substrate combination: glutamate+malate 6-Month-old (n=8) 24-Month-old (n=8) P 
State 3 (nmol of O/min per mg of protein) 159.9±14.5 139.6±15.3 0.0165 
State 4 (nmol of O/min per mg of protein) 28.2±2.8 22.4±3.1 0.0015 
RCR 5.7±0.6 6.3±0.7 0.0870 
High ADP (2 mM concentration) (nmol of O/min per mg of protein) 215.0±17.9 187.5±24.2 0.0216 
Uncoupled rate (nmol of O/min per mg of protein) 213.5±27.3 190.8±24.2 0.1448 
ADP/O ratio 2.84±0.23 2.76±0.18 0.4514 
Substrate combination: glutamate+malate 6-Month-old (n=8) 24-Month-old (n=8) P 
State 3 (nmol of O/min per mg of protein) 159.9±14.5 139.6±15.3 0.0165 
State 4 (nmol of O/min per mg of protein) 28.2±2.8 22.4±3.1 0.0015 
RCR 5.7±0.6 6.3±0.7 0.0870 
High ADP (2 mM concentration) (nmol of O/min per mg of protein) 215.0±17.9 187.5±24.2 0.0216 
Uncoupled rate (nmol of O/min per mg of protein) 213.5±27.3 190.8±24.2 0.1448 
ADP/O ratio 2.84±0.23 2.76±0.18 0.4514 

A panel of substrates, including complexes I, II, III and IV, and fatty acid substrates, were used to evaluate the rates of OXPHOS in intact mitochondrial preparations isolated from the kidneys of 6- and 24-month-old animals. The rates of State 3 respiration were slower in 24-month-old animals for the substrates glutamate plus malate, α-oxoglutarate plus malonate and DHQ, and the fatty acid substrates (Figure 1). The rate of State 3 respiration in the presence of high ADP (a concentration of 2 mM) was significantly slower in mitochondria obtained from 24-month-old animals for all substrates tested, except for two of the complex I substrates, glutamate and the combination of pyruvate and malate (Figure 1).

OXPHOS rates in 6- and 24-month-old kidney mitochondria

Figure 1
OXPHOS rates in 6- and 24-month-old kidney mitochondria

(A) Substrates for polarographic assays of OXPHOS through complex I (glutamate), complex II (succinate), complex III (DHQ) and complex IV (TMPD) are shown. ADP-stimulated (2 mM) rates of State 3 respiration were determined in freshly isolated mitochondria from 6-month-old (light grey bars, n=8) and 24-month-old (dark grey bars, n=8) rats. Uncoupled rates determined after the addition of DNP are shown for substrates: glutamate, succinate and DHQ. (B) ADP-stimulated (2 mM) rates of State 3 respiration are shown for the substrate combinations: pyruvate and malate (P+M), α-oxoglutarate (αKG) and malonate, acetyl-carnitine and malate (AC+M), palmitoyl-carnitine and malate (PC+M) and palmitoyl-CoA with malate (PCoA+M) and carnitine. Values are means±S.D. for each experimental group. *P<0.05 and †P<0.01, calculated using a non-paired two-tailed Student's t test.

Figure 1
OXPHOS rates in 6- and 24-month-old kidney mitochondria

(A) Substrates for polarographic assays of OXPHOS through complex I (glutamate), complex II (succinate), complex III (DHQ) and complex IV (TMPD) are shown. ADP-stimulated (2 mM) rates of State 3 respiration were determined in freshly isolated mitochondria from 6-month-old (light grey bars, n=8) and 24-month-old (dark grey bars, n=8) rats. Uncoupled rates determined after the addition of DNP are shown for substrates: glutamate, succinate and DHQ. (B) ADP-stimulated (2 mM) rates of State 3 respiration are shown for the substrate combinations: pyruvate and malate (P+M), α-oxoglutarate (αKG) and malonate, acetyl-carnitine and malate (AC+M), palmitoyl-carnitine and malate (PC+M) and palmitoyl-CoA with malate (PCoA+M) and carnitine. Values are means±S.D. for each experimental group. *P<0.05 and †P<0.01, calculated using a non-paired two-tailed Student's t test.

In order to determine whether the defects observed in OXPHOS were attributable to intrinsic deficits of the respiratory complexes, the enzyme activities of the ETC were assayed in freshly isolated mitochondria. We did not detect a significant difference in the activities of any of the components of the ETC (Supplementary Figure S1 at http://www.BiochemJ.org/bj/427/bj4270105add.htm). Consistent with this observation, the protein abundance of the individual ETC complexes was similar between the two groups (Supplementary Figures S2A, S2C, S2E and S2G at http://www.BiochemJ.org/bj/427/bj4270105add.htm). In order to determine whether the abundance of the ETC supercomplexes differed between the two groups, mitochondria were solubilized with digitonin and separated using blue native gel electrophoresis (Supplementary Figure S2B). There were no significant differences in the protein abundance of supercomplexes, S0, S1 or S2, between the two groups (Supplementary Figures S2D, S2F and S2H).

The morphology of mitochondria isolated from a 6-month-old and 24-month-old animal was evaluated by electron microscopy (Figure 2). Isolated mitochondria were predominantly of a proximal and distal tubule origin. There were no obvious morphological differences between the 6- and 24-month-old mitochondrial preparations.

Transmission electron micrographs of mitochondria isolated from kidneys of 6- and 24-month-old Fischer 344 rats

Figure 2
Transmission electron micrographs of mitochondria isolated from kidneys of 6- and 24-month-old Fischer 344 rats

Mitochondria were isolated from 6-month-old (A) and 24-month-old (B) animals using differential centrifugation, and fixed for transmission electron microscopy. Scale bars=1.0 μm.

Figure 2
Transmission electron micrographs of mitochondria isolated from kidneys of 6- and 24-month-old Fischer 344 rats

Mitochondria were isolated from 6-month-old (A) and 24-month-old (B) animals using differential centrifugation, and fixed for transmission electron microscopy. Scale bars=1.0 μm.

The content of cytochromes aa3, b, c1 and c were assayed using a spectrophotometric method to determine whether differences in the cytochromes could be contributing to the decrement in OXPHOS capacity seen in the mitochondria of 24-month-old animals. We found a 23% and 37% decrease in the abundance of cytochrome b and cytochrome c respectively, in mitochondria isolated from the kidneys of 24-month-old animals (Figure 3A). The abundance of cytochrome aa3 and cytochrome c1 was not significantly different between the two groups (Figure 3A). Immunoblotting confirmed a decrease in cytochrome c in the mitochondria isolated from 24-month-old animals relative to 6-month-old animals (Figure 3C). The abundance of cytochrome c was decreased by 31% by band densitometry in mitochondria isolated from the 24-monthold animals compared with the 6-month-old controls (Figure 3B). The decrease in cytochrome c was not related to differences in mitochondrial loading or loss of integrity of the OMM (outer mitochondrial membrane) as the abundance of VDAC, a marker of the OOM, and Smac/Diablo, a soluble protein in the intermembranous space, was unchanged between the two groups (Supplementary Figure S3 at http://www.BiochemJ.org/bj/427/bj4270105add.htm).

Determination of cytochrome content in 6- and 24-month-old RKM

Figure 3
Determination of cytochrome content in 6- and 24-month-old RKM

RKM from 6- and 24-month-old animals (n=7 for each group) were solubilized with deoxycholate and the cytochrome content was determined spectrophotometrically using the difference spectra (A). Values are means±S.D. for each experimental group. Cytochrome c was detected using a monoclonal antibody on immunoblots of RKM isolated from 6- and 24-month-old animals (n=4 for each group) separated by SDS/PAGE and transferred on to PVDF membranes (C). The molecular mass in kDa is indicated in the centre of the blot. Band densitometry confirmed a significant decrease in cytochrome c content in RKM isolated from 24-month-old animals (B). *P<0.05, calculated using non-paired, two-tailed Student's t test.

Figure 3
Determination of cytochrome content in 6- and 24-month-old RKM

RKM from 6- and 24-month-old animals (n=7 for each group) were solubilized with deoxycholate and the cytochrome content was determined spectrophotometrically using the difference spectra (A). Values are means±S.D. for each experimental group. Cytochrome c was detected using a monoclonal antibody on immunoblots of RKM isolated from 6- and 24-month-old animals (n=4 for each group) separated by SDS/PAGE and transferred on to PVDF membranes (C). The molecular mass in kDa is indicated in the centre of the blot. Band densitometry confirmed a significant decrease in cytochrome c content in RKM isolated from 24-month-old animals (B). *P<0.05, calculated using non-paired, two-tailed Student's t test.

In order to confirm the functional relevance of the decrease in the cytochrome c abundance observed in the mitochondria of 24-month-old animals, we performed a polarographic assay of COX on permeabilized mitochondria without and with the addition of 16 nmol of exogenous cytochrome c. Without the addition of exogenous cytochrome c, the rate of oxygen consumption through COX in mitochondria isolated from 24-month-old animals was 645 nmol of O/min per mg of mitochondrial protein, 23% slower than the rate in 6-month-old animals (836 nmol of O/min per mg of mitochondrial protein) and was highly significant (P=0.002) (Figure 4). This difference was corrected by the addition of exogenous cytochrome c.

Polarographic COX assay

Figure 4
Polarographic COX assay

RKM from 6-month-old (n=7) and 24-month-old (n=7) animals were permeabilized and COX activity was evaluated by the rate of oxygen consumption after the addition of TMPD and ascorbate with endogenous cytochrome c (COX) and with the addition of exogenous cytochrome c (COX+CytC). Values are means±S.D. for each experimental group. *P<0.005, calculated using a non-paired, two-tailed Student's t test.

Figure 4
Polarographic COX assay

RKM from 6-month-old (n=7) and 24-month-old (n=7) animals were permeabilized and COX activity was evaluated by the rate of oxygen consumption after the addition of TMPD and ascorbate with endogenous cytochrome c (COX) and with the addition of exogenous cytochrome c (COX+CytC). Values are means±S.D. for each experimental group. *P<0.005, calculated using a non-paired, two-tailed Student's t test.

DISCUSSION

In the present study we show that RKM develop a defect in OXPHOS with age (Figure 1). Assays of OXPHOS capacity in intact mitochondria test not only the activities of the ETC complexes, but also substrate transporters and dehydrogenases, membrane integrity and elements which transfer electrons between ETC complexes (i.e. coenzyme Q and cytochrome c). Our use of a comprehensive panel of substrates to test the integrated mitochondrial function in OXPHOS assays has permitted the biochemical dissection of the pathway and allowed the site of the defect to be localized.

Our data demonstrate an age-related OXPHOS defect with the use of substrates, which enter the ETC at complexes I, II, III and cytochrome c, and is not relieved by the addition of an uncoupler demonstrating the site of the defect to occur at cytochrome c or complex IV. Variance in the degree of decline in State 3 rates between different metabolic substrates suggests that substrate transporters and dehydrogenases may also have a contributory role in the age-related decline of substrate utilization for OXPHOS within the kidney. The first-order rate constant of complex IV was unchanged in spectrophotometric assays of solubilized mitochondria from aged kidney in the presence of exogenous reduced cytochrome c (Supplementary Figure S1), arguing that an intrinsic defect in complex IV is not the principal cause of the diminished OXPHOS capacity in mitochondria isolated from 24-month-old animals.

mtDNA deletions and concomitant loss of ETC activity has been convincingly shown to occur in an age-dependent fashion in the rat kidney [8]. The principle defect in OXPHOS related to mtDNA deletions should be a decrease in activity of the respiratory complexes containing subunits encoded on the mtDNA (i.e. complexes I, III and IV). We found no significant changes in the respiratory complexes with spectrophotometric measurement of activity or blue native gel assessment of protein abundance (Supplementary Figures S1 and S2). Therefore our results do not support a defect in the respiratory complexes as the principal cause of the reduction in OXPHOS observed and suggest that mtDNA deletions are not responsible for the OXPHOS defect in these studies. This may not be surprising given the observation that the ETC-deficient renal tubular cells only comprised ~0.2% of the cortical volume at 24 months [8], and mitochondria derived from these tubules would therefore be expected to make a relatively minor contribution to the total mitochondrial population.

Therefore we sought alternative mechanisms, including a decrease in mitochondrial cytochrome c content, to explain the agedependent defects in OXPHOS that we have observed. The cytochrome b and cytochrome c content in mitochondria isolated from kidneys of 24-month-old animals was 23% and 37% lower respectively (Figure 3). The total cytochrome b content is distributed between complexes II and III within the mitochondria [24]. We judged the decline in cytochrome b to be non-limiting, as the activities of complex II (coenzyme Q reductase) and complex III (ubiquinol-cytochrome c oxidoreductatase) were no different between the two groups (Supplementary Figure S1). The fact that the cytochrome b content is divided between the two complexes may attenuate the effect a reduction in the total cytochrome b content has on either complex individually and may contribute to the lack of functional differences in these complexes between the two groups. This suggested that the observed decline in OXPHOS capacity of the 24-month-old RKM was due to the decreased cytochrome c content. To test this hypothesis we performed polarographic assays of COX, complex IV, without and with exogenous cytochrome c. The addition of exogenous cytochrome c corrected the defect in the polarographic activity of cytochrome c oxidase. This demonstrates that the depletion of cytochrome c is the dominant mechanism for the decrease in OXPHOS capacity observed in mitochondria obtained from the kidneys of 24-month-old animals (Figure 4).

The observed decrease of cytochrome c content in mitochondria isolated from kidneys of 24-month-old animals is not explained by leak of cytochrome c across the OMM, as we demonstrated that the OMM was impermeable to cytochrome c prior to addition of the detergent dodecyl maltoside in the spectrophotometric assay of COX activity (Supplementary Figure S1). It is known that the transcription of cytochrome c declines with age in human and murine kidneys [25,26]. This leads to the hypothesis that an age-related decline in cytochrome c transcription underlies the reduction in mitochondrial cytochrome c abundance, which we have observed in aged animals. However, a transcriptional microarray experiment in rat kidney failed to identify age-related changes in cytochrome c transcription [27] and alternative explanations, including increased rates of cytochrome c turnover or decreased capacity for cytochrome c binding to the inner mitochondrial membrane must also be considered as possibilities. The age-related transcriptional decline of cytochrome c has not been described for extra-renal organs, and the reasons underlying this tissue-specific finding have yet to be examined.

Modulation of flux through the ETC in direct relation to mitochondrial cytochrome c content, independent of the activities and protein abundance of the respiratory complexes, is not completely novel. Serum-stimulated murine fibroblasts demonstrated an increase in oxygen consumption related to an increase in cytochrome c content [28]. Furthermore, fibroblasts isolated from individuals with early onset Parkinson's disease and mutations in PINK1 were demonstrated to have decreased respiratory capacity related to a decrease in cytochrome c content [29]. Cultured neurons derived from 24-month-old rat brains appear to have reduced cytochrome c content and complex IV activity when compared with 9-month-old controls [30].

The decline in OXPHOS efficiency is demonstrated by a decrease in State 3 and State 4 rates. Thus there is not only a slower rate of ADP consumption, implying a slower rate of ATP production, in mitochondria isolated from 24-month-old animals, there is also a lower rate of proton leak across the inner mitochondrial membrane or a decline in ATPase activity unassociated with the F1Fo ATPase. Interestingly, the integrity of the mitochondria, as reflected by the RCR, and coupling to the phosphorylation apparatus, as reflected by the ADP/O ratios, in renal mitochondria appear to be preserved with aging in the kidney. This result is contrary to a previous report in which both the RCR and ADP/O ratios were decreased in mitochondria isolated from the kidneys of 22-month-old Wistar rats compared with 4-month-old controls [9], but agrees with two others [31,32] demonstrating preservation of renal mitochondrial RCR and ADP/O ratios with aging.

The results of the present study demonstrate an age-related mitochondrial defect in the pool of mitochondria obtained from whole kidney. Future efforts will be needed to determine whether the decline in OXPHOS shown here is a generalized phenomenon in renal mitochondria or if it localizes to mitochondria isolated from a defined nephron segment. The nephron travels a circuitous route through the cortex and medulla and is composed of multiple segments often containing multiple cell types. Renal mitochondria are not distributed evenly in all cell types, with the highest density of mitochondria found in the medullary and cortical thick ascending limb of Henle's loop followed by the proximal tubule [33]. In addition, the kidney is able to metabolize a broad array of substrates for energy production and substrate preferences have been shown to vary along the length of the nephron [34]. Finally, the renal microenvironment varies dramatically in a radial fashion from the cortex to the medulla with increasing tonicity and decreasing oxygen tension.

From these studies we can conclude that the mitochondrial content per g of kidney weight is lower in 24-month-old animals compared with 6-month-old animals. The mitochondria isolated from the elderly animals demonstrate a decrease in OXPHOS capacity mediated by decreases in State 3 and State 4 respiration, which could not be explained by decrements in the biochemical activities of the individual components of the ETC or the protein abundance of the ETC protein complexes. The mitochondrial content of cytochrome c is decreased in mitochondria isolated from the kidneys of 24-month-old animals. The decrease in cytochrome c significantly contributes to the decreased rate of oxygen consumption by COX observed in the mitochondria of 24-month-old animals. The decline in cytochrome c and COX activity results in a global decrease in OXPHOS capacity, as all of the OXPHOS substrates tested require electron flux through cytochrome c and COX.

Abbreviations

     
  • COX

    cytochrome c oxidase

  •  
  • DHQ

    duroquinol

  •  
  • ETC

    electron transport chain

  •  
  • HRP

    horseradish peroxidase

  •  
  • mtDNA

    mitochondrial DNA

  •  
  • OMM

    outer mitochondrial membrane

  •  
  • OXPHOS

    oxidative phosphorylation

  •  
  • RCR

    respiratory control ratio

  •  
  • RKM

    rat kidney mitochondria

  •  
  • ROS

    reactive oxygen species

  •  
  • TBS

    Tris-buffered saline

  •  
  • TMPD

    N,N,N′,N′-tetramethyl-p-phenylenediamine

  •  
  • VDAC

    voltage-dependent anion channel

AUTHOR CONTRIBUTION

Charles Hoppel and John O'Toole contributed to the experimental design, data analysis and manuscript preparation. Hiral Patel and Colin Naples performed the citrate synthase and ETC assays. Hisashi Fujioka performed the electron microscopy experiments. John O'Toole completed all other experimental methods.

We would like to gratefully acknowledge the support and technical advice provided by David Kehres, William Parland, Edwin Vazquez and Mariana Rosca.

FUNDING

This work was supported by the National Institutes of Health [grant numbers DK071108 (to J.F.O.), PO1AG015885 (to C.L.H.)]; and the Kidney Foundation of Ohio (to J.F.O.).

References

References
1
Davies
D. F.
Shock
N. W.
Age changes in glomerular filtration rate, effective renal plasma flow, and tubular excretory capacity in adult males
J. Clin. Invest.
1950
, vol. 
29
 (pg. 
496
-
507
)
2
Lindeman
R. D.
Tobin
J.
Shock
N. W.
Longitudinal studies on the rate of decline in renal function with age
J. Am. Geriatr. Soc.
1985
, vol. 
33
 (pg. 
278
-
285
)
3
Zhang
Q. L.
Rothenbacher
D.
Prevalence of chronic kidney disease in population-based studies: systematic review
BMC Public Health
2008
, vol. 
8
 pg. 
117
 
4
Lameire
N.
Van
B. W.
Vanholder
R.
The changing epidemiology of acute renal failure
Nat. Clin. Pract. Nephrol.
2006
, vol. 
2
 (pg. 
364
-
377
)
5
Gullans
S. R.
Brenner
B. M.
Metabolic basis of solute transport
The Kidney
2000
Philadelphia
W.B. Saunders Company
(pg. 
215
-
246
)
6
Wallace
D. C.
A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine
Annu. Rev. Genet.
2005
, vol. 
39
 (pg. 
359
-
407
)
7
Guarente
L.
Mitochondria: a nexus for aging, calorie restriction, and sirtuins?
Cell
2008
, vol. 
132
 (pg. 
171
-
176
)
8
McKiernan
S. H.
Tuen
V. C.
Baldwin
K.
Wanagat
J.
Djamali
A.
Aiken
J. M.
Adult-onset calorie restriction delays the accumulation of mitochondrial enzyme abnormalities in aging rat kidney tubular epithelial cells
Am. J. Physiol. Renal Physiol.
2007
, vol. 
292
 (pg. 
F1751
-
F1760
)
9
de Cavanagh
E. M.
Piotrkowski
B.
Basso
N.
Stella
I.
Inserra
F.
Ferder
L.
Fraga
C. G.
Enalapril and losartan attenuate mitochondrial dysfunction in aged rats
FASEB J.
2003
, vol. 
17
 (pg. 
1096
-
1098
)
10
Meng
Q.
Wong
Y. T.
Chen
J.
Ruan
R.
Age-related changes in mitochondrial function and antioxidative enzyme activity in fischer 344 rats
Mech. Ageing Dev.
2007
, vol. 
128
 (pg. 
286
-
292
)
11
Sachan
D. S.
Hoppel
C. L.
Carnitine biosynthesis. Hydroxylation of N6-trimethyl-lysine to 3-hydroxy-N6-trimethyl-lysine
Biochem. J.
1980
, vol. 
188
 (pg. 
529
-
534
)
12
DiMarco
J. P.
Hoppel
C.
Hepatic mitochondrial function in ketogenic states. Diabetes, starvation, and after growth hormone administration
J. Clin. Invest.
1975
, vol. 
55
 (pg. 
1237
-
1244
)
13
Chance
B.
Williams
G. R.
Respiratory enzymes in oxidative phosphorylation. III. The steady state
J. Biol. Chem.
1955
, vol. 
217
 (pg. 
409
-
427
)
14
Estabrook
R. W.
Mitochondrial respiratory control and polarographic measurement of ADP/O ratios
Methods Enzymol.
1967
, vol. 
10
 (pg. 
41
-
47
)
15
Matsuoka
Y.
Srere
P. A.
Kinetic studies of citrate synthase from rat kidney and rat brain
J. Biol. Chem.
1973
, vol. 
248
 (pg. 
8022
-
8030
)
16
Hoppel
C. L.
Kerr
D. S.
Dahms
B.
Roessmann
U.
Deficiency of the reduced nicotinamide adenine dinucleotide dehydrogenase component of complex I of mitochondrial electron transport. Fatal infantile lactic acidosis and hypermetabolism with skeletal-cardiac myopathy and encephalopathy
J. Clin. Invest.
1987
, vol. 
80
 (pg. 
71
-
77
)
17
Palmer
J. W.
Tandler
B.
Hoppel
C. L.
Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle
J. Biol. Chem.
1977
, vol. 
252
 (pg. 
8731
-
8739
)
18
Sordahl
L. A.
McCollum
W. B.
Wood
W. G.
Schwartz
A.
Mitochondria and sarcoplasmic reticulum function in cardiac hypertrophy and failure
Am. J. Physiol.
1973
, vol. 
224
 (pg. 
497
-
502
)
19
Hoppel
C.
Cooper
C.
The action of digitonin on rat liver mitochondria. The effects on enzyme content
Biochem. J.
1968
, vol. 
107
 (pg. 
367
-
375
)
20
Krahenbuhl
S.
Talos
C.
Wiesmann
U.
Hoppel
C. L.
Development and evaluation of a spectrophotometric assay for complex III in isolated mitochondria, tissues and fibroblasts from rats and humans
Clin. Chim. Acta
1994
, vol. 
230
 (pg. 
177
-
187
)
21
Williams
J. N.
Jr
A method for the simultaneous quantitative estimation of cytochromes a, b, c1, and c in mitochondria
Arch. Biochem. Biophys.
1964
, vol. 
107
 (pg. 
537
-
543
)
22
Wittig
I.
Braun
H. P.
Schagger
H.
Blue native PAGE
Nat. Protoc.
2006
, vol. 
1
 (pg. 
418
-
428
)
23
Sternberg
S. R.
Biomedical image processing
IEEE Computer
1983
, vol. 
16
 (pg. 
22
-
34
)
24
Benard
G.
Faustin
B.
Passerieux
E.
Galinier
A.
Rocher
C.
Bellance
N.
Delage
J. P.
Casteilla
L.
Letellier
T.
Rossignol
R.
Physiological diversity of mitochondrial oxidative phosphorylation
Am. J. Physiol. Cell Physiol.
2006
, vol. 
291
 (pg. 
C1172
-
C1182
)
25
Rodwell
G. E.
Sonu
R.
Zahn
J. M.
Lund
J.
Wilhelmy
J.
Wang
L.
Xiao
W.
Mindrinos
M.
Crane
E.
Segal
E.
, et al. 
A transcriptional profile of aging in the human kidney
PLoS Biol.
2004
, vol. 
2
 pg. 
e427
 
26
Zahn
J. M.
Sonu
R.
Vogel
H.
Crane
E.
Mazan-Mamczarz
K.
Rabkin
R.
Davis
R. W.
Becker
K. G.
Owen
A. B.
Kim
S. K.
Transcriptional profiling of aging in human muscle reveals a common aging signature
PLoS Genet.
2006
, vol. 
2
 pg. 
e115
 
27
Chen
G.
Bridenbaugh
E. A.
Akintola
A. D.
Catania
J. M.
Vaidya
V. S.
Bonventre
J. V.
Dearman
A. C.
Sampson
H. W.
Zawieja
D. C.
Burghardt
R. C.
Parrish
A. R.
Increased susceptibility of aging kidney to ischemic injury: identification of candidate genes changed during aging, but corrected by caloric restriction
Am. J. Physiol. Renal Physiol.
2007
, vol. 
293
 (pg. 
F1272
-
F1281
)
28
Herzig
R. P.
Scacco
S.
Scarpulla
R. C.
Sequential serum-dependent activation of CREB and NRF-1 leads to enhanced mitochondrial respiration through the induction of cytochrome c
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
13134
-
13141
)
29
Piccoli
C.
Sardanelli
A.
Scrima
R.
Ripoli
M.
Quarato
G.
D'Aprile
A.
Bellomo
F.
Scacco
S.
De
M. G.
Filla
A.
, et al. 
Mitochondrial respiratory dysfunction in familiar parkinsonism associated with PINK1 mutation
Neurochem. Res.
2008
, vol. 
33
 (pg. 
2565
-
2574
)
30
Jones
T. T.
Brewer
G. J.
Critical age-related loss of cofactors of neuron cytochrome C oxidase reversed by estrogen
Exp. Neurol.
2009
, vol. 
215
 (pg. 
212
-
219
)
31
Weinbach
E. C.
Garbus
J.
Coenzyme A content and fatty acid oxidation in liver and kidney mitochondria from aged rats
Gerontologia
1959
, vol. 
3
 (pg. 
253
-
260
)
32
Gold
P. H.
Gee
M. V.
Strehler
B. L.
Effect of age on oxidative phosphorylation in the rat
J. Gerontol.
1968
, vol. 
23
 (pg. 
509
-
512
)
33
Pfaller
W.
Rittinger
M.
Quantitative morphology of the rat kidney
Int. J. Biochem.
1980
, vol. 
12
 (pg. 
17
-
22
)
34
Wirthensohn
G.
Guder
W. G.
Renal substrate metabolism
Physiol. Rev.
1986
, vol. 
66
 (pg. 
469
-
497
)

Supplementary data