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Background: A low-protein diet supplemented with ketoacids (LPD + KA) maintains the nutritional status of patients with chronic kidney disease (CKD). Oxidative damage and mitochondrial dysfunction associated with the upregulation of p66SHC and FoxO3a have been shown to contribute to muscle atrophy. This study aimed to determine whether LPD + KA improves muscle atrophy and attenuates the oxidative stress and mitochondrial damage observed in CKD rats. Methods: 5/6 nephrectomy rats were randomly divided into three groups and fed with either 22% protein (normal-protein diet; NPD), 6% protein (low-protein diets; LPD) or 5% protein plus 1% ketoacids (LPD + KA) for 24 weeks. Sham-operated rats with NPD intake were used as the control. Results: KA supplementation improved muscle atrophy and function in CKD + LPD rats. It also reduced the upregulation of genes related to the ubiquitin-proteasome system and 26S proteasome activity, as well as protein and mitochondrial oxidative damage in the muscles of CKD + LPD rats. Moreover, KA supplementation prevented the drastic decrease in activities of mitochondrial electron transport chain complexes, mitochondrial respiration, and content in the muscles of CKD + LPD rats. Furthermore, KA supplementation reversed the elevation in p66Shc and FoxO3a expression in the muscles of CKD + LPD rats. Conclusions: Our results showed that KA supplementation to be beneficial to muscle atrophy in CKD + LPD, which might be associated with improvement of oxidative damage and mitochondrial dysfunction through suppression of p66Shc and FoxO3a. Keywords: Chronic kidney disease, Muscle atrophy, Ketoacids, Oxidative stress, Mitochondrial dysfunction Background development of malnutrition including muscle wasting Dietary protein restriction is one of the major compo- as a result of restricted-protein diets. Ketoacids (KA), a nents of therapy for patients with chronic kidney disease nitrogen-free ketoanalogue, can reduce endogenous urea (CKD). It can minimise uremic symptoms and slow the formation, toxic ions, and metabolic products in the progression of renal failure . However, there has been CKD model . Moreover, KA can provide a sufficient increasing concern regarding the risk of subsequent amount of essential amino acids to maintain nutritional status in CKD patients . Therefore, it has been pre- scribed together with low-protein diets (LPD) to patients * Correspondence: email@example.com with advanced CKD. Despite the large number of studies Dongtao Wang and Lianbo Wei contributed equally to this work. Department of Traditional Chinese Medicine, Shenzhen Hospital, Southern on low-protein diets with ketoacids (LPD + KA) that Medical University, Shenzhen 518000, Guangdong, China have been performed to improve muscle atrophy in 5/6 Department of Nephrology, Shenzhen Traditional Chinese Medicine nephrectomy and type 2 diabetic nephropathy rat Hospital, Guangzhou University of Traditional Chinese Medicine, Shenzhen 518033, Guangdong, China models [4–6], the mechanism of its preventive effects on Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Skeletal Muscle (2018) 8:18 Page 2 of 13 oxidative damage and mitochondrial dysfunction in skel- against CKD-induced oxidative damage and mitochon- etal muscle of CKD remains unclear. drial dysfunction by mediating the p66Shc and FoxO3a Oxidative stress is involved in the pathogenesis of a signalling in the muscles of 5/6 nephrectomised rats. number of chronic diseases, such as CKD, cancer, chronic heart failure, and diabetes mellitus. In the set- Methods ting of CKD, oxidative damage is a known cause of Animal experiments muscle atrophy, which can contribute to muscle dys- Sprague-Dawley male rats (obtained from the Experimen- function and mark myofibrillar proteins for degradation tal Animal Centre of the Southern Medical University, . The imbalance between increased production of China, certification no. SCXK (Yue) 2006–0015) weighing reactive oxygen species (ROS) and limited antioxidant 180–220 g were housed in a room at a constant capacity can lead to severe damage on cellular compo- temperature with a 12-h light–12-h dark cycle and were nents such as DNA, proteins, nucleic acids, and lipids in given free access to food and water. These rats were kept pathological conditions . Hydrogen peroxide (H O ) according to the guidelines of Care and Use of Laboratory 2 2 is a major component of ROS, generated during mito- Animals formulated by the Ministry of Science and chondrial respiration, which induced mitochondrial dys- Technology of China, and all experimental procedures function in skeletal muscle . Concurrently, oxidants were approved by the Ethics Committee of the Southern may stimulate the pathways of skeletal muscle protein Medical University. Male rats were randomly assigned to degradation, such as the ubiquitin-proteasome system either the 5/6 nephrectomy group or the sham-operated (UPS) . Mammalian cells have a sophisticated system group. Each animal in the nephrectomy group underwent for scavenging ROS to non-toxic forms to defend cells 5/6 nephrectomy by the ablation of two-thirds mass of the against oxidative stress induced by high levels of ROS. left kidneys and subsequent right unilateral nephrectomy This antioxidant defence system is composed of antioxi- after 1 week. In a sham-operated rat, a sham operation dant enzymes such as superoxide dismutase (SOD) and was performed. Then, 1 week after the operation, the 5/6 catalase . So far, there is evidence that ROS gener- nephrectomy group was randomly separated and main- ation in mitochondria is critical for mitochondrial tained on three different diets: a normal-protein diet (22% dysfunction and decreased mitochondrial content, which protein, NPD), a low-protein diet (6% protein, LPD) or a are controlled by regulating signal transduction, gene ex- LPD supplemented with KA (5% protein and 1% KA, LPD pression, and redox reaction [12, 13]. Among these, + KA). The sham group with a normal-protein diet acted p66Shc has been shown to contribute to mitochondrial as the control. Each group included ten rats. These diets ROS (mtROS) production by sequestering electrons were fed to the groups for a period of 24 weeks. By the from the respiratory chain to regulate its redox function end of the study, one rat in NPD group had died, but all within mitochondria [14, 15]. In addition, FoxO3a is a the other rats survived. member of the FoxO family of proteins, which has been implicated in initiating protein degradation during Experimental diets muscle atrophy . It has been reported that FoxO3a Rats were fed either an NPD, LPD or LPD + KA diet. reduces ROS generation by the transcriptional activation KA was provided by Beijing Fresenius Kabi Pharmaceut- of SOD and catalase [17, 18]. However, the role of ical Company Limited. The KA composition was as fol- p66Shc and FoxO3a in mediating oxidative stress in the lows (mg/630 mg): racemic keto isoleucine, 67 mg; skeletal muscle of CKD has not been reported. ketones leucine, 101 mg; phenylalanine ketone, 68 mg; Mitochondrial dysfunction plays a pivotal role in the ketones valine, 86 mg; DL-methionine hydroxy, 59 mg; pathology of muscle atrophy induced by disuse, cancer, lysine acetate, 105 mg; threonine, 53 mg; tryptophan, and ageing [19–21]. We have previously shown that mito- 23 mg; histidine, 38 mg; tyrosine, 30 mg; total N, 36 mg. chondrial dysfunction, characterised by mitochondrial loss The three diets were formulated according to the Ameri- and sedentary dynamics, plays a key role in CKD-induced can Institute of Nutrition for Rodent Diets, AIN-93, and muscle atrophy . In addition, previous clinical and ani- all had the same energy content (15.7 kJ/g (3.8 kcal/g)), mal studies have shown that CKD disrupts mitochondrial vitamins and minerals. The details of the composition of morphology and oxidative capacity, which subsequently the three diets are as described in our previous study causes oxidative damage . However, the role of oxida- . The animals in all groups had free access to food tive stress and mitochondrial dysfunction in CKD-induced and water provided ad libitum. muscle atrophy is a controversial topic. In the present study, we aimed to investigate the role of ROS generation Grip power and running distance with p66Shc and FoxO3a signalling and antioxidant en- Grip power was measured using a dynamometer for rats zymes on skeletal muscle mitochondrial dysfunction in (ZH-YLS-13A, Anhui Zhenghua Biological Instrument CKD rats. We hypothesised that LPD + KA protects Equipment Co., Ltd., Huaibei, China). A rat was put on Wang et al. Skeletal Muscle (2018) 8:18 Page 3 of 13 a metal bar and pulled horizontally. The power of traction was used for the determination of quadriceps muscle when the rat released the metal bar was defined as the content of MDA by colorimetric method as previously grip power. Running distance was measured using a tread- described , using a protein carbonyl colorimetric mill for mice (Yuyan Instrument Co., Ltd., Shanghai, assay kit. The result was expressed as nanomoles per China), in accordance with a previously described protocol milligram protein. . Rats were made to run on the motor-driven treadmill until they were exhausted, which was defined as the point Determination of lipid hydroperoxides at which they remained on the electrical shocker plate A modified ferrous oxidation-xylenol (FOX) orange (mild stimulation of 0.2 mA, equivalent to a medically technique was performed to measure the lipid hydroper- used electric therapy equipment) at the end of the tread- oxides in accordance with a previously described mill for more than 30 s. The treadmill was set at a 10% in- protocol . Briefly, quadriceps muscles were homoge- cline; the speed was 30 cm/s at the beginning and was nised (1:4 wt/vol) in potassium phosphate buffer increased by 3 cm/s every 2 min. The average running (50 mM, pH 7.8) and centrifuged at 12,000g for 15 min time until exhaustion was approximately 40 min. at 4 °C. For this assay, the pellet was discarded and 20 μl of the supernatant (250 μg of protein) was mixed with Biochemical parameters FOX reagent (250 μM ammonium ferrous sulphate, After 24 weeks of treatment, the rats were sacrificed by 100 μM xylenol orange and 25 mM H SO ) at a final 2 4 sodium pentobarbital and blood samples were subse- volume of 200 μl. It was incubated at room temperature quently collected. Serum biochemical indexes serum for 30 min. The absorbance of the samples was read at creatinine (Scr), blood urea nitrogen (BUN) and albumin 560 nm. (ALB) were detected using a Roche automatic biochem- ical analyser. Detection of ROS generation by dihydroethidium fluorescence staining Morphological studies (HE, SDH staining) and Dihydroethidium (DHE) oxidation products were de- measurement of myofiber size and SDH activity tected as described previously . Quadriceps muscles The tibialis anterior (TA) muscle samples were sectioned were cut into 10-μm-thick sections and were incubated and stained with haematoxylin and eosin (HE) and with DHE (5 μM) in PBS in a light-protected incubator succinate dehydrogenase (SDH, complex II of the re- at 37 °C for 30 min. They were washed with PBS to spiratory chain) in line with standard procedures. Myofi- remove excess DHE and then mounted. The fluores- ber cross-sectional area (CSA) and SDH activity were cence was evaluated in a confocal microscope (Zeiss determined as previously reported . LSM510Meta). Laser excitation at 488 nm and emission at 610 nm were used. The detection was made using a Assay of 26S proteasome activity 560-nm long-pass filter. ImageJ (NIH) software was Chymotrypsin-like activity of proteasome was assayed applied to quantitatively analyse the fluorescent images. using the fluorogenic peptide (LLVY-MCA, Enzo Life The results were shown as arbitrary units of Sciences item #P802–0005) as described previously . fluorescence. Assays were carried out in a microtiter plate by diluting 25 mg of cytosolic protein into 200 mL of 10 mM Muscle mitochondrial isolation MOPS, pH 7.4 containing 25 mM LLVY-MCA (sub- Mitochondrial isolation from the skeletal muscle was 2+ strate), 25 mM ATP and 5.0 mM Mg . Rate of fluores- modified from the protocol described by Boutagy . cent product formation was measured with excitation Briefly, red muscle was removed from the quadriceps and emission wavelengths of 350 and 440 nm, respect- muscle and finely minced with scissors. The muscle was ively. Peptidase activities were measured in the absence then transferred to 10 ml mitochondrial homogenate and presence (20 mM) of the proteasome-specific inhibi- buffer in tissue homogeniser. The muscle was homoge- tor epoxomicin and the difference between the two rates nised using a motorised pestle and kept on ice at all was attributed to the proteasome. times. After homogenisation, the homogenate buffer was gently poured into a 15-ml centrifuge tube and centri- Determination of malondialdehyde (MDA) activity and fuged at 1300 g for 5 min at 4 °C. The supernatant was carbonyl content absorbed onto a mitochondrial centrifugation buffer in A part of the homogenate was used for the determin- an overspeed centrifuge tube and centrifuged at 17,000 g ation of quadriceps muscle activity of MD, and was de- for 10 min at 4 °C. Soon after, the sediment was trans- termined according to the method described by Buege ferred into 9 ml of isolation buffer in another pre-chilled and Aust . The results were expressed as nanomoles overspeed centrifuge tube. This was centrifuged at per milligram protein. The other part of the homogenate 10,000 g for 10 min at 4 °C. The sediment was Wang et al. Skeletal Muscle (2018) 8:18 Page 4 of 13 transferred to a new pre-chilled 1.5-ml microcentrifuge (Jiancheng Biotech Inc., Nanjing, China) in strict accord- tube and re-suspended in 1 ml of isolation buffer. This ance with the manufacturer’s instructions. The adduct was centrifuged at 8000 g for 10 min at 4 °C. The super- was measured spectrophotometrically at 550 nm with a natant was removed, and the sediment was gently mixed plate reader (TECAN infinite M200, USA). Similarly, the with 300 μl of storage buffer. The concentration of mito- activity of catalase (U/mg protein) in the mitochondria chondrial protein was determined with a BCA Protein of the gastrocnemius muscles was measured using a Assay kit (Pierce, Rockford, IL, USA) and the protein commercial kit (Jiancheng Biotech Inc., Nanjing, China), yields of IFM were calculated accordingly. which is based on the reaction of ammonium molybdate with H O to form a light-yellow complex compound. 2 2 Oxygen consumption rate (OCR) Adduct was measured spectrophotometrically at 405 nm OCR was measured using the Seahorse XF 24 Extracel- with a plate reader (TECAN infinite M200, USA) in lular Flux Analyzers (Seahorse Bioscience, Billerica, MA, strict accordance with the manufacturer’s instructions. USA), as described previously . Ten micrograms of mitochondria (3 to 6 μl) were loaded at the centre of the Quantitative real-time PCR XF24 cell culture microplates (Seahorse Bioscience) on Total RNA was isolated from the quadriceps muscles ice, and 50 μl of the substrates (5 mM pyruvate plus using Trizol (Invitrogen, Carlsbad, CA). RNA concentra- 5 mM malate) and 440 μl of mitochondrial assay solu- tion and integrity were assessed. cDNA was synthesised tion (MAS) (70 mM sucrose, 220 mM mannitol, 5 mM using iScript cDNA Synthesis Kit at 70 °C for 10 min, KH PO , 5 mM MgCl , 2 mM HEPES, 1 mM EGTA, followed by incubation at 42 °C for 60 min and at 95 °C 2 4 2 and 0.2% BSA, pH 7.4) were carefully added on top. All for 10 min. The genes analysed were p66Shc, FoxO3a, the chemicals loaded in the Seahorse cartridge ports Atrogin-1, MuRF-1, MUSA1, C5 and C2 proteasome were diluted in MAS (pH = 7.4). subunits and GAPDH (reference gene) (Table 1). All primers were synthesised by Invitrogen. Quantitative Measurement of electron transport chain activity real-time PCR was run for all genes separately, and All assays were performed at 30 °C with a Shimadzu amplifications were performed by the ABI Prism 5700 UV-1601 spectrophotometer. The specific activities of Sequence Detection System (Applied Biosystems) using complexes I, II, III and IV were assayed as described by SYBR Green PCR Master Mix (Applied Biosystems). Sundaram Kumaran et al. . Results were quantified as Ct values, where Ct is defined as the threshold cycle of the polymerase chain reaction Determination of mitochondrial H O content, SOD and at which the amplified product is first detected. Expres- 2 2 catalase activity sion was normalised by GAPDH levels as an endogenous The hydrogen peroxide content in the skeletal muscle reference. Sham group levels were arbitrarily set at 1. mitochondria was measured by the colorimetric method as previously described , using a commercial kit, Western blotting based on the reaction with molybdic acid (Jiancheng Snap-frozen quadriceps muscle tissues were homogenised Biotech Inc., Nanjing, China). Adduct was measured in lysis buffer as previously reported. Cytosolic and mito- spectrophotometrically at 405 nm in a plate reader chondrial proteins were separated on a 10% SDS-PAGE (TECAN infinite M200, USA) in strict accordance with gel and then transferred to a PVDF membrane (Bio-Rad the manufacturer’s instructions. The total SOD activity Laboratories, Hercules, CA, USA). The membrane’s (U/mg protein) in the mitochondria of the gastrocne- non-specific binding sites were blocked at 26 °C for 1 h mius muscles was measured using a commercial kit with 5% non-fat milk powder in Tris-buffered saline/ Table 1 Primer sequences Gene Forward Reverse P66SHC 5′-TACAACCCACTTCGGAATGGTCT-3′ 5′-ATGTACCGAACCAAGTAGG-3′ FoxO3a 5′-CAGGTGTGTGCTGCTATGAACATC-3′ 5′-GTCTTCGTGCTCGGTGATG-3′ C5 subunit 5′-GCTGCTCGACAACCAGGTTGGCTTC-3′ 5′-CAGTGTACACATCCCTCTCGGCTGCAG-3′ C2 subunit 5′-TTGAAGAAAGACCACAGAGAAAAGCACAGC-3′ 5′-GTATGCCCCTGCATCCTCATGTCCTC-3′ Atrogin-1 5′-TACTAAGGAGCGCCATGGATACT-3′ 5′-GTTGAATCTTCTGGAATCCAG GAT-3′ MuRF1 5′-GTGTGAGGTGCCTACTTGCT-3′ 5′-ACTCAGCTCCTCCTTCACCT-3′ MUSA1 5′-ACCACGACCCTGATGATGAGC-3′ 5′-GGTCAGGCTCTTCCATTCGTCT-3′ GAPDH 5′-GTTCAACGGCACAGTCAAGG-3′ 5′-GTGGTGAAGACGCCAGTAGA-3′ Wang et al. Skeletal Muscle (2018) 8:18 Page 5 of 13 Tween-20 (TBST) and then incubated overnight at Table 2 Renal function data (means ± SD) 4 °C with primary antibodies. After washing with Group Scr(μmol/l) BUN(mmol/l) ALB(g/l) TBST, the membranes were incubated with secondary Sham 60.61 ± 22.80 7.21 ± 2.12 89.40 ± 17.25 antibodies for 1 h at room temperature with shaking. NPD 168.11 ± 43.18*** 17.01 ± 3.32*** 60.84 ± 12.21** After washing, protein bands were detected and ana- †† † †† LPD 141.61 ± 36.67*** 12.01 ± 7.78** 45.16 ± 10.89*** lysed using a ChemiDoc™ MP Imaging System ††‡ ††‡ ‡ LPD + KA 135.59 ± 25.66*** 9.08 ± 1.89* 59.67 ± 5.66** (Bio-Rad Laboratories, CA, USA). VDAC and GAPDH Sham normal-protein diet, NPD normal-protein diet, LPD low-protein diet, LPD were used as the loading controls for mitochondrial +KA low-protein diet supplemented with KA, Scr serum creatinine BUN blood protein and cytosolic protein, respectively. Results were urea nitrogen, ALB albumin. Mean value was significantly different from that of the sham group: *P < 0.05, expressed as the integrated optical density relative to **P < 0.01,***P < 0.001 VDAC or GAPDH. SOD1 (1:1000, SAB2500976) was ob- Mean value was significantly different from that of the NPD group: † †† tained from Sigma-Aldrich (Diegem, Belgium). VDAC P < 0.05, P < 0.01 Mean value was significantly different from that of the LPD group: P < 0.05 (1:1000, #4661) and FoxO3a (1:1000, #2497) were obtained from Cell Signaling Technologies (Danvers, MA, USA). p66Shc (1:200, sc-1695) was obtained from Santa Cruz Biotechnology (CA, USA). GAPDH (1:1000, 60004-1-Ig) was digitorum longus (EDL) muscle masses of the NPD and obtained from Proteintech (Chicago, IL, USA). LPD groups were lower than those of the sham group. However, KA supplementation partially decreased the Statistical analysis muscles mass losses compared to the LPD group (Fig. 1b). Results are shown as the mean ± SD. One-way analysis of Furthermore, the improved muscle mass in the LPD + KA variance (ANOVA) followed by the Student-Newman-Keuls group was confirmed by an increase in the mean test was used to compare the differences between the means cross-sectional area of the TA muscle in the LPD group in more than two groups. The level of significance was set at (Fig. 1c, d). The grip power was lower in the NPD and P < 0.05. All the statistical analyses were performed with LPD group than in the sham group. However, the de- SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA). ceased grip power was partially corrected with KA supple- mentation in the LPD + KA group. There was no Results difference between the NPD and LPD groups (Fig. 1e). Ketoacid supplementation preserves renal function in Similarly, the running distance of the NPD and LPD CKD + LPD rats groups was significantly reduced, while KA supplementa- At the end of the study, the 5/6 nephrectomy group dis- tion triggered an increase compared to the LPD group. played significantly higher Scr and BUN levels compared On the other hand, the LPD + KA group also showed an with the sham group. The level of Scr was found to be increase in the running distance compared to the NPD highest in the NPD group, but significantly decreased in group (Fig. 1f). the LPD group and lowest in the LPD + KA group. Meanwhile, BUN levels were highest in the NPD group and significantly reduced in the LPD group; the LPD + Ketoacid supplementation reduces the upregulation of KA group had the lowest values. On the other hand, genes related to the ubiquitin-proteasome system and serum ALB levels were lower in the 5/6 nephrectomy 26S proteasome activity in the muscles of CKD + LPD rats groups than the sham group. Among the 5/6 nephrec- The 5/6 nephrectomy groups exhibited a significant in- tomy groups, the LPD group had lower serum ALB duction of mRNA expression in Atrgin-1 and MuRF1 levels than the NPD and LPD + KA groups, but no stat- compared to the sham group; however, KA supplemen- istical differences were observed between the NPD and tation reduced the levels of Atrgin-1 and MuRF1 mRNA LPD + KA groups (Table 2). in the LPD + KA group compared to the LPD group (Fig. 2a, b). In addition, the LPD group displayed an in- Ketoacid supplementation improves muscle atrophy and crease in the expression of MUSA1 mRNA, and this function in CKD + LPD rats change was abolished by KA intervention (Fig. 2c). The body weight of the sham group was significantly Moreover, the levels of C5 proteasome subunit mRNA higher than that of the 5/6 nephrectomy group. Among and 26S chymotrypsin-like proteasome activity were the 5/6 nephrectomy group, body weight was found to significantly increased in the NPD and LPD groups be the lowest in the LPD group and significantly in- compared with the sham group, and this change of creased in the LPD + KA group. No statistical differences the LPD group was also abolished by KA supplemen- were observed between the NPD and LPD + KA groups tation (Fig. 2e, f). However, no changes were observed (Fig. 1a). Moreover, the quadriceps (Quad), gastrocne- in C2 proteasome subunit mRNA levels among all mius (Gastroc), tibialis anterior (TA) and extensor the groups (Fig. 2d). Wang et al. Skeletal Muscle (2018) 8:18 Page 6 of 13 Fig. 1 Body weight, muscle mass, muscle fibre cross-sectional area (CSA) and muscle function in the experimental groups. a Body weight changes. b Weights of quadriceps (Quad), gastrocnemius (Gastroc), tibialis anterior (TA) and extensor digitorum longus (EDL) muscles normalised by tibia length. c Cross sections of tibialis anterior (TA) muscle stained with haematoxylin and eosin (HE). Scale bar 50 μm. d Muscle fibre CSA (mm ) of TA muscle. e Grip power. d Running distance. Results are presented as the mean ± SD, n = 6 per group. Mean value was significantly different from that of the sham group: *P < 0.05, **P < 0.01. Mean value was significantly different from that of the NPD group: †P < 0.05, ††P < 0.01. Mean value was significantly different from that of the LPD group: ‡P < 0.05. Sham, normal-protein diet; NPD, normal-protein diet; LPD, low-protein diet; LPD + KA, low-protein diet supplemented with ketoacids Fig. 2 The ubiquitin-proteasome pathway in skeletal muscle of the experimental groups. a Atrogin-1, b MuRF1, c MUSA1, d C5 subunit and e C2 subunit mRNA levels, and f 26S chymotrypsin-like proteasome activity. Results are presented as the mean ± SD, n = 6 per group. Mean value was significantly different from that of the sham group: *P < 0.05, **P < 0.01. Mean value was significantly different from that of the NPD group: †P < 0.05, ††P < 0.01. Mean value was significantly different from that of the LPD group: ‡P < 0.05. Sham, normal-protein diet; NPD, normal-protein diet; LPD, low-protein diet; LPD + KA, low-protein diet supplemented with ketoacids Wang et al. Skeletal Muscle (2018) 8:18 Page 7 of 13 Ketoacid supplementation suppresses oxidative stress in Moreover, the mitochondrial catalase activity was the muscles of CKD + LPD rats slightly reduced in the NPD and LPD groups, and these Muscle atrophy in CKD is associated with increased oxi- changes appeared to be partially reversed with KA sup- dative stress. Therefore, the markers of ROS, such as plementation, as these differences did not reach signifi- H O , MDA, lipid hydroperoxide, carbonyl content and cance (Fig. 4b). Furthermore, mitochondrial SOD 2 2 DHE oxidation products, were detected. The NPD and activity and SOD1 protein were decreased in the NPD LPD groups showed higher levels of H O (Fig. 3a), and LPD groups compared to those in the sham group. 2 2 MDA (Fig. 3b), lipid hydroperoxide (Fig. 3c), carbonyl In addition, the LPD group showed a significant increase content (Fig. 3d) and DHE oxidation products (Fig. 3e, in mitochondrial SOD activity compared to the NPD f) in skeletal muscle compared to the sham group. group (Fig. 4c). Interestingly, the decreased mitochon- Moreover, a low protein diet also caused a slight in- drial SOD activity and SOD1 protein in the LPD group crease in the markers of ROS compared to the NPD was prevented by KA supplementation (Fig. 4c, d). group, although these changes were not statistically significant except for MDA levels. The addition of KA to Ketoacid supplementation increases the activity of LPD prevented the overexpression of these ROS mitochondrial electron transport chain complexes in the markers. muscles of CKD + LPD rats To determine whether CKD induces mitochondrial dys- Ketoacid supplementation improves mitochondrial function, we firstly determined the activities of mito- oxidative capacity in the muscles of CKD + LPD rats chondrial electron transport chain complexes in To analyse the consequences of KA supplementation on gastrocnemius muscle. The activities of mitochondrial mitochondrial oxidative capacity, we evaluated the levels complexes I, II, III and IV were significantly decreased of mitochondrial H O , catalase and SOD in the skeletal in the 5/6 nephrectomy groups compared to those in the 2 2 muscle of the experimental rats. The NPD and LPD sham group (Fig. 5a–d). Among the 5/6 nephrectomy groups displayed an increase in the mitochondrial H O groups, the activities of mitochondrial complexes I and 2 2 content compared with the sham group, while KA sup- IV were found to be the lowest in the NPD group, and plementation decreased the elevation of mitochondrial significantly higher in the LPD and LPD + KA groups; H O compared to the LPD and NPD groups (Fig. 4a). however, the LPD + KA group showed a significantly 2 2 Fig. 3 The marks of oxidative stress in skeletal muscle of the experimental groups. Quantification of skeletal muscle a H O , b MDA, c lipid 2 2 hydroperoxides and d carbonyl contents. e Immunofluorescence staining for dihydroethidium (DHE). Scale bar = 100 μm. f Quantification of DHE intensity. Results are presented as the mean ± SD, n = 6 per group. Mean value was significantly different from that of the sham group: *P < 0.05, **P < 0.01. Mean value was significantly different from that of the NPD group: †P < 0.05, ††P < 0.01. Mean value was significantly different from that of the LPD group: ‡P < 0.05. Sham, normal-protein diet; NPD, normal-protein diet; LPD, low-protein diet; LPD + KA, low-protein diet supplemented with ketoacids Wang et al. Skeletal Muscle (2018) 8:18 Page 8 of 13 Fig. 4 Mitochondrial oxidative capacity in skeletal muscle of the experimental groups. a Mitochondrial H O content, b mitochondrial catalase 2 2 activity, c mitochondrial SOD activity and d mitochondrial SOD1 protein. Results are presented as the mean ± SD, n = 6 per group. Mean value was significantly different from that of the sham group: *P < 0.05, **P < 0.01. Mean value was significantly different from that of the NPD group: †P < 0.05, ††P < 0.01. Mean value was significantly different from that of the LPD group: ‡P < 0.05. Sham, normal-protein diet; NPD, normal-protein diet; LPD, low-protein diet; LPD + KA, low-protein diet supplemented with ketoacids Fig. 5 Activities of mitochondrial electron transport chain complexes in skeletal muscle of the experimental groups. The activity of a mitochondrial complex I (NADH-coenzyme Q oxidoreductase), b mitochondrial complex II (succinate dehydrogenase-coenzyme Q oxidoreductase), c mitochondrial complex III (coenzyme Q cytochrome c oxidoreductase) and d mitochondrial complex IV (cytochrome c oxidase) from skeletal muscle of the experimental rats. Results are presented as the mean ± SD, n = 6 per group. Mean value was significantly different from that of the sham group: *P < 0.05, **P < 0.01. Mean value was significantly different from that of the NPD group: †P < 0.05, ††P < 0.01. Mean value was significantly different from that of the LPD group: ‡P < 0.05. Sham, normal-protein diet; NPD, normal-protein diet; LPD, low-protein diet; LPD + KA, low-protein diet supplemented with ketoacids Wang et al. Skeletal Muscle (2018) 8:18 Page 9 of 13 higher level of activity compared to the LPD group prominent in the LPD + KA group than in the LPD (Fig. 5a, d). In addition, the NPD and LPD groups group (Fig. 6d). showed significantly lower levels of activity in mitochon- drial complexes II and III compared to the sham group Ketoacid supplementation decreases expression of and were not statistically significant between the NPD p66Shc and FoxO3a proteins in the muscles of CKD + LPD and LPD groups. As expected, the LPD + KA group rats exhibited reduced levels of activity in mitochondrial Evidence indicates that p66shc serves as a redox enzyme complexes II and III compared to the LPD and NPD and has been implicated in mitochondrial ROS gener- groups (Fig. 5b, c). ation and translation of oxidative signals. The expression of p66Shc protein and mRNA was significantly higher in Ketoacid supplementation improves the mitochondrial the NPD and LPD groups, respectively, compared to that content and function in the muscles of CKD + LPD rats in the sham group. However, KA treatment decreased Mitochondrial content was assessed using SDH stain- the level of the p66Shc protein and mRNA compared to ing of TA muscles (Fig. 6a). The SDH stain intensity the level in the LPD group. Moreover, LPD and LPD + was markedly reduced in the NPD and LPD groups KA groups displayed a lower level of p66Shc protein and compared to that in the sham group, but distinctly mRNA than the NPD group (Fig. 7a, c). On the other increased in the LPD + KA group compared to that in hand, an upward trend in FoxO3a protein expression the LPD group (Fig. 6b). Moreover, the results and mRNA in the NPD and LPD groups was also ob- showed that the mitochondrial yield of intermyofibril- served in comparison to the sham group, but KA sup- lar mitochondria (IFM) was significantly lower in the plementation induced a decrease compared to the LPD NPD and LPD groups compared to that in the sham group. In addition, the LPD and LPD + KA groups also group, but showed remarkable improvement with KA showed a lower level of the FoxO3a protein and mRNA treatment in the LPD + KA group compared to that than the NPD group (Fig. 7b, d). in the LPD group (Fig. 6c). Consistently, the basal mitochondrial respiration ability (oxygen consumption Discussion rate, OCR) of IFM was decreased in the NPD and These experiments provide novel insights into the mech- LPD groups compared to that in the sham group. anisms responsible for KA supplementation playing a Furthermore, the increase in OCR was also more protective role in muscle atrophy and its function in Fig. 6 Mitochondrial content and mitochondrial oxygen consumption rate (OCR) in skeletal muscle of the experimental groups. a SDH staining was performed on 10-μm-thick sections from gastrocnemius muscles frozen in liquid nitrogen-cooled isopentane. Scale bar 50 μm. b Quantification of SDH-stain intensity (expressed in A.U). c Mitochondrial yield in isolated quadriceps muscles. d The mitochondrial oxygen consumption rate (OCR) in isolated quadriceps muscles. Results are presented as the mean ± SD, n = 6 per group. Mean value was significantly different from that of the sham group: *P <0.05, **P < 0.01. Mean value was significantly different from that of the NPD group: †P <0.05, ††P < 0.01. Mean value was significantly different from that of the LPD group: ‡P < 0.05. Sham, normal-protein diet; NPD, normal-protein diet; LPD, low-protein diet; LPD + KA, low-protein diet supplemented with ketoacids Wang et al. Skeletal Muscle (2018) 8:18 Page 10 of 13 Fig. 7 Expression of p66Shc and FoxO3a mRNA, and proteins in the skeletal muscle of the experimental groups. a p66Shc and b FoxO3a mRNA expression was measured by RT-PCR and is presented as corrected for GAPDH and normalised to the sham group. c Upper: representative immunoblotting of p66Shc and GAPDH. Lower: the ratio of p66Shc and GAPDH normalised to the sham group. d Upper: representative immunoblotting of FoxO3a and GAPDH. Lower: the ratio of FoxO3a and GAPDH normalised to theshamgroup.Results are presented as the mean ± SD, n = 6 per group. Mean value was significantly different from that of the sham group: *P < 0.05, **P < 0.01. Mean value was significantly different from that of the NPD group: †P < 0.05, ††P < 0.01. Mean value was significantly different from that of the LPD group: ‡P < 0.05. Sham, normal-protein diet; NPD, normal-protein diet; LPD, low-protein diet; LPD + KA, low-protein diet supplemented with ketoacids CKD-LPD rats. In the present study, the data indicate proteins appear to mediate in skeletal muscle catabolism: that CKD-LPD-induced loss of muscle mass and func- Atrogin1/MAFbx, MuRF1 and MUSA1. Atrogin1 and tion is attributed to a significant reduction in fibre CSA MuRF1 are upregulated in a number of catabolic condi- compared to sham rats. Surprisingly, these changes were tions including cancer, diabetes, kidney failure and sepsis reversed by KA supplementation. Our findings are in . Indeed, the expression of Atrogin-1, MuRF1 and agreement with our own previous studies as well as MUSA1 were increased in CKD-LPD rats. Moreover, those conducted by other researchers [4–6], highlighting these increases correlate with increased C5 subunit CKD-LPD-dependent fibre atrophy as the primary cause mRNA and 26S proteasome activity. Importantly, these of muscle mass loss in advancing CKD . Moreover, changes were prevented by KA supplementation, which our findings confirm that KA supplementation sup- is consistent with our results from previous works . presses UPS activation and protects skeletal muscle from Collectively, the present results indicate that KA supple- oxidative damage in CKD + LPD rats. Furthermore, our mentation plays a muscle-protective role in CKD-LPD, results show that KA supplementation prevents de- at least in part, via inhibition of the UPS. creases in the activity of mitochondrial electron trans- Considerable evidence has indicated that muscle atro- port chain complexes and increases mitochondrial phy with CKD has been linked to an altered oxidative respiration and content in the muscles of CKD + LPD status of redox-sensitive proteins , and increased rats. Furthermore, our findings confirm that KA supple- oxidative modifications of virtually all cellular macro- mentation reduces the production of mtROS, and molecules, including lipids, DNA and proteins . p66Shc and FoxO3a expression in the muscles of CKD Muscle atrophy can be exacerbated by oxidative stress, + LPD rats. which promotes the production of reactive carbonyl It is widely accepted that the UPS is the main route by compounds and lipoperoxides leading to the accumula- which proteins are degraded during muscle atrophy. tion of advanced glycation and lipoxidation end products This involves the targeted degradation of proteins via . In the present study, our data showed that modification by ubiquitin and subsequent proteolysis by CKD-LPD-induced loss of muscle mass and function is the 26S proteasome . Proteins targeted by ubiquitin associated with increased oxidative damage including an are modified through the actions of three types of increase in H O and MDA levels, accumulation of car- 2 2 ubiquitin-conjugating enzymes: E1, E2 and E3. Three E3 bonyl content and DHE oxidation products and Wang et al. Skeletal Muscle (2018) 8:18 Page 11 of 13 increased levels of lipid peroxidation, suggesting that CKD-associated decline in mitochondrial function , changes in redox homeostasis toward an oxidised state the effect of mitochondrial dysfunction and mtROS as may be a contributor to skeletal muscle atrophy. Previ- the underlying key regulators of the CKD-related atro- ous work from our own group  and others [5, 6] have phy process remains unclear. The present study shows shown that KA supplementation has a beneficial that the activities of mitochondrial complexes I, II, III anti-atrophy effect in CKD animals, which is consistent and IV were found to be significantly decreased in the with our results. Furthermore, we found that the marks skeletal muscle of CKD-LPD rats. To our surprise, KA of oxidative stress were decreased in skeletal muscle supplementation improved the activity of complexes I, when supplemented with KA compared to LPD alone. II, III and IV in the skeletal muscle of CKD-LPD rats. Genetic manipulations of redox regulatory systems were Several studies suggest that oxidative damage to mito- found to modify the muscle atrophy process . Col- chondrial DNA may be responsible for the decrease in lectively, the present results indicate that KA supple- the activity of electron transport chain enzyme com- mentation ameliorates oxidative damage in the skeletal plexes in aged rats and amyotrophic lateral sclerosis muscle of CKD + LPD rats. mouse models [45, 46]. Oxidative damage to proteins is Skeletal muscle has a high mitochondrial content, and associated with numerous alterations in mitochondrial skeletal muscle mitochondria have been reported to ex- respiratory capacity and amount. In the present study, hibit an increase in mtROS in disuse  and ageing we found that the mitochondrial content and the rate of [39, 40]. In the present study, we have shown that iso- oxygen consumption were significantly decreased in the lated skeletal muscle mitochondria from CKD + LPD rats skeletal muscle of CKD-LPD rats. Furthermore, KA exhibit an increase in H O generation, which was con- supplementation can effectively reverse mitochondrial 2 2 sistent with the role of mitochondria as a contributor to respiratory capacity and mitochondrial loss in the LPD CKD-related muscle oxidative damage . Moreover, + KA group. Evidence suggests that mitochondrial dys- treatment with KA protects against CKD-LPD-induced function plays a key role in the pathology of muscle at- increases in mitochondrial H O content, which may se- rophy induced by CKD [44, 47]. Ourselves as well as 2 2 lectively protect mitochondria from oxidative damage. other researchers have previously reported that On the other hand, increased oxidative stress arises from CKD-induced dominant mitochondrial dysfunction, an imbalance between pro-oxidant and antioxidant fac- characterised by mitochondrial loss, compromised mito- tors and is depicted in skeletal muscle under catabolic or chondrial respiration, and disrupted mitochondrial dis- dysfunctional conditions . In the present study, our tribution and morphology [22, 47]. Collectively, the results show that the CKD-LPD group displays a present results indicate that KA supplementation in- decrease in mitochondrial catalase activity, SOD activity, creases muscle mitochondrial mass and the activity of and protein expression in skeletal muscle, which was mitochondrial electron transport chain enzyme com- reversed by KA supplementation. Importantly, while plexes and improves mitochondrial respiration in CKD mitochondria-targeted antioxidant ameliorates muscle + LPD rats. loss and mitochondrial dysfunction of skeletal muscle Following these findings, we investigated several in ageing rats , targeted overexpression of intracellular signalling pathways mediating mitochon- mitochondrial catalase protects against cancer drial ROS that could contribute to muscle wasting. chemotherapy-induced skeletal muscle atrophy and Specifically, we focused on p66Shc and FoxO3a dysfunction . Therefore, strong evidence of redox activation because both are activated by oxidant stress imbalance-induced skeletal muscle atrophy supports and both contribute to muscle wasting [48, 49]. our hypothesis that mitochondrial oxidative damage is Recent studies also implicate p66Shc in a redox-dependent a major determinant of skeletal muscle loss in CKD. pathway that sensitises cells to proapoptotic stimuli In addition, KA treatment reduces mitochondrial ROS by activating AKT, phosphorylating FoxO transcrip- and provides clear antioxidant protective effects on tion factors and preventing the induction of antioxi- muscle atrophy in CKD + LPD rats. dant/free radical scavenging genes . Our findings Skeletal muscle atrophy with CKD is associated with confirm that upregulation of p66Shc and FoxO3a mitochondrial dysfunction including the decrease of expression in the skeletal muscle of CKD-LPD rats activity in mitochondrial electron transport chain en- and, importantly, treatment with KA decreases the zyme complexes and mitochondrial content, and re- expression of both p66Shc and FoxO3a in CKD + duced mitochondrial respiratory capacity [41, 44]. The LPD rats. These results strongly suggest that the mitochondrial respiratory chain is a powerful source of p66Shc-FoxO3a pathway plays a role in the regulation ROS, considered as a potential mechanism contributing of mitochondrial ROS production and muscle oxida- to mitochondrial dysfunction. Although cumulative tive stress responses, and that this pathway may oxidative damage has been suggested to induce mediate the anti-oxidative effects of KA. Wang et al. Skeletal Muscle (2018) 8:18 Page 12 of 13 Conclusions 2. 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Skeletal Muscle – Springer Journals
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