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Fasting reduces oxidative stress, mitochondrial dysfunction and fibrosis induced by renal ischemia-reperfusion injury
Free Radical Biology and Medicine 135 (2019) 60–67
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Original article
Fasting reduces oxidative stress, mitochondrial dysfunction and fibrosis induced by renal ischemia-reperfusion injury
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Pedro Rojas-Moralesa, Juan Carlos León-Contrerasb, Omar Emiliano Aparicio-Trejoa, Jazmin Gabriela Reyes-Ocampoa, Omar Noel Medina-Camposa, Angélica Saraí Jiménez-Osorioa, Susana González-Reyesa, Brenda Marquina-Castillob, Rogelio Hernández-Pandob, Diana Barrera-Oviedoc, Laura Gabriela Sánchez-Lozadad, José Pedraza-Chaverria, Edilia Tapiad,∗ a
Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico Departamento de Patología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Ciudad de México 14080, Mexico Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico d Laboratorio de Fisiopatología Renal, Departamento de Nefrología, Instituto Nacional de Cardiología Ignacio Chávez, Ciudad de México 14080, Mexico b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Fasting Oxidative stress Mitochondrial dysfunction Fibrosis Ischemia-reperfusion injury Acute kidney injury Chronic kidney disease
Food deprivation protects against ischemia-reperfusion (IR) injury through unknown mechanisms. In an experimental rat model of acute IR injury, we found that preoperative fasting for 3 days protects rats from tubular damage and renal functional decline by increasing antioxidant protection independently of the NF-E2-related factor 2 (Nrf2), and by maintaining mitochondrial morphology and function. In addition, further analysis revealed that fasting protects against tubulointerstitial fibrosis. In summary, our results point out to fasting as a robust nutritional intervention to limit oxidative stress and mitochondrial dysfunction in early acute kidney injury and also to promote long-term protection against fibrosis.
1. Introduction Dietary restriction (DR) and fasting are nutritional interventions strongly associated with longevity and acute stress resistance in a disparate range of model organisms [1,2]. For example, both DR and fasting regimens delay age-related chronic diseases and also enhance intracellular defense against chemotherapy and traumatic injury in multiple tissues [2–4]. However, the underlying molecular mechanisms remain largely unknown. Early studies have shown that preoperative fasting protects mice against renal ischemia-reperfusion (IR) injury [5,6], a stressful situation that notably impairs kidney structure and function, often leading to chronic kidney disease (CKD) and high mortality in the clinic [7]. As in the seminal paper by Mitchell et al. [5] was reported that fasting increases the messenger ribonucleic acid (mRNA) of certain antioxidant proteins in IR injury, this work was rapidly embraced by the scientific community as evidence of the protective effect of fasting against IRinduced oxidative stress [4]. However, to our knowledge, no experimental evidence exists to prove this assumption. Given that oxidative stress and also mitochondrial dysfunction are the major contributing factors in the pathophysiology of IR injury
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[8–10], we investigated in the current work whether the protective effect of fasting is associated with maintenance of redox balance, mitochondrial function and morphology in a rat model of ischemic acute kidney injury (AKI). Additionally, we explored whether fasting confers long-lasting protection against kidney fibrosis, a maladaptive repair process after IR injury driving CKD progression [11]. 2. Materials and methods 2.1. Reagents The following reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA): Adenosine 5′-diphosphate sodium salt (ADP), antimycin A, horseradish peroxidase (HRP), bovine serum albumin (BSA) fatty acid free (A6003), BSA (A4503) 1-chloro-2,4 dinitrochlorobenzene (CDNB), carbonyl cyanide m-chlorophenylhydrazone (CCCP), sodium succinate dibasic, sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic (NaH2PO4), sodium glutamate, sodium malate, K-lactobionate, manganese (II) chloride (MgCl2) tetrahydrate, rotenone, ethylenediaminetetraacetic acid (EDTA), protease inhibitor cocktail, ethylene glycol-bis(2-aminoethylether)-
Corresponding author. E-mail address: [email protected] (E. Tapia).
https://doi.org/10.1016/j.freeradbiomed.2019.02.018 Received 23 November 2018; Received in revised form 1 February 2019; Accepted 16 February 2019 Available online 25 February 2019 0891-5849/ © 2019 Elsevier Inc. All rights reserved.
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N,N,N′,N′-tetraacetic acid (EGTA), glutamate, glutaraldehyde, glutathione (GSH), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), malate, D-mannitol, sodium chloride (NaCl), nicotinamide adenine dinucleotide phosphate (NADPH), sodium fluoride (NaF), sodium orthovanadate (Na3VO4), nitroblue tetrazolium (NBT), Nonidet P40 (NP-40), paraformaldehyde, phenylmethylsulfonyl fluoride (PMSF), safranin O, sodium deoxycholate, sodium dodecyl sulfate (SDS), sucrose, taurine, Tris-HCl, xanthine, xanthine oxidase, dinitrophenylhydrazine, streptomycin sulfate, guanidine hydrochloride and primary antibodies against tubulin (#T9026). Ethanol, hydrochloric acid, ethyl acetate, and trichloroacetic acid were from JT Baker (Xalostoc, Mexico). Antibodies against kidney injury molecule 1 (KIM-1) were from R&D systems (Minneapolis, MN, USA). Antibodies against 3-nitrotyrosine (3-NT, #9691) were from Cell Signaling (Danvers, MA, USA). Primary antibodies against 4-hydroxynoneal (4-HNE, #AB5605) were from Merck Millipore (Burlington, MA, USA). Primary antibodies against NF-E2-related factor 2 (Nrf2, #sc-722) were from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies against cytochrome c oxidase subunit 1 (MTCO1) [1D6E1A8] (#ab14705), cytochrome b-c1 complex subunit 2 (UQCRC2) [13G12AF12BB11] (#ab14745), and heat shock protein 60 (HSP60, #ab31115) were from Abcam (Cambridge, MA, USA). Primary antibodies against alpha smooth muscle actin (α-SMA, #GTX100034) were from GeneTex (Irvine, CA, USA). The IRDye® secondary antibodies were acquired from LI-COR Biosciences (Lincoln, NE, USA). Commercial kits to measure blood urea nitrogen (BUN) and creatinine levels in plasma were obtained from SpinReact (Girona, Spain). The Bio-Rad protein assay dye reagent concentrate to measure total protein (Bradford assay) was from Bio Rad Laboratories Inc. (Hercules, CA, USA). The following kits RNeasy, Omniscript reverse transcriptase (RT) and QuantiTect SYBR Green polymerase chain reaction (PCR) were form Qiagen (Germantown, MD, USA).
Fig. 1. Experimental designs. (A) To investigate the protective effect of fasting, rats were either fed ad libitum or fasted for 3 days before ischemia-reperfusion (IR) injury. 24 h after IR, rats were euthanized to analyze renal damage, oxidative stress markers and mitochondrial function. At 28 days post IR injury, rats were euthanized to analyze fibrosis development. (B) To analyze Nrf2 protein expression in the liver and kidney rats were fasted for 1–3 days without IR injury.
deposition (Fig. 1). 2.4. Renal function analysis Creatinine and BUN levels were assessed in plasma by commercial kits according to manufacturer's instructions. For proteinuria determination, rats were housed individually in metabolic cages and urine was collected over a 24-h period. Urinary protein excretion was measured by the Bradford assay [14].
2.2. Experimental animals
2.5. Protein carbonyls
Male Wistar rats (8–9 weeks old, n = 5–6 per group) weighing 240–260 g were housed in standard laboratory conditions. Rats were divided in three groups: (a) Sham, (b) fasted (beginning at 10 a.m.) for 3 days before IR injury, (c) fed before IR injury. An additional group of rats was fasted during 1, 2 or 3 days and euthanized to obtain kidneys and livers to measure Nrf2 levels. Experiments were approved by the Animal care committee (CICUAL) of the National Institute of Cardiology “Ignacio Chávez” (#18–1050).
For protein carbonylation determination, tissue extracts were derivatized with 10 mM 2,4-dinitrophenylhydrazine for 1 h and then proteins were precipitated with 20% trichloroacetic acid. After washing for several times with an ethanol-ethyl acetate mixture (1:1 v/v), proteins were solubilized in 6 M guanidine hydrochloride and the absorbance was recorded at 370 nm [15].
2.3. IR injury model
Enzyme activities were assessed in kidney lysates as previously described [16]. Briefly, GST activity was assessed by following the formation of the GSH-CDNB adduct at 340 nm. SOD activity was measured at 560 nm as the inhibition of NBT reduction in a superoxidegenerating system by xanthine-xanthine oxidase. GPx activity was measured by following NADPH disappearance at 340 nm in a spectrophotometer in a GSH-regenerating system [16].
2.6. Enzyme assays
An established rat model of AKI was used, induced by unilateral IR injury plus contralateral nephrectomy, as previously described [12,13]. Briefly, under sodium pentobarbital anesthesia (60 mg/kg, i.p.), the right kidney was excised and ischemia was done in the left kidney by clamping the renal artery for 30 min, using non-traumatic vascular clips. For sham surgery, the kidneys were exposed but the renal artery was not clamped. After surgery, rats were fed ad libitum and sacrificed 24 h or 4 weeks later to harvest blood and the remaining kidney for analysis (Fig. 1). The following parameters were measured in the group of 24 h: markers of biochemical and structural renal damage (plasma levels of creatinine and BUN, renal KIM-1 levels and kidney histological studies by light microscopy), activity of the antioxidant enzymes glutathione S-transferase (GST), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in kidney lysates, markers of oxidant stress (protein carbonyls, 4-HNE, and 3-NT), renal Nrf2 levels, mRNA levels of Nrf2, heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase-1 (NQO-1), markers of mitochondrial function (oxygen consumption, mitochondrial membrane potential, mitochondrial ultrastructure and mitonuclear protein imbalance). Renal fibrosis was measured in the group of 4 weeks by measuring α-SMA levels and fibrous collagen
2.7. Mitochondrial function To evaluate mitochondrial function, kidney mitochondria were isolated by differential centrifugation in cold (4 °C) isolation buffer (225 mM D-mannitol, 75 mM sucrose, 1 mM EDTA, 5 mM HEPES, 0.1% BSA, pH 7.4) as previously described [16]. The final mitochondria pellet was resuspended in 100 μL of BSA-free isolation buffer and the mitochondrial total protein was measured by the Lowry method. Isolated mitochondria (200 μg of total protein) were loaded into the chamber that contained 2 ml of MiR05 respiration buffer: 0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose and 1 g/L BSA essentially fatty acid free. Mitochondrial oxygen consumption rate during state 3 (ADP-stimulated, 2.5 mM) and state 4°, induced by 2.5 μM oligomycin 61
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Fig. 2. Protective effect of fasting against biochemical and structural renal damage induced by renal ischemia-reperfusion (IR). Rats were either fed ad libitum or fasted for 3 days before IR injury. Plasma levels of (A) blood urea nitrogen (BUN) and (B) creatinine, and (C) urinary protein excretion assessed 24 h after IR. (D) Representative hematoxylin and eosin (H&E)-stained kidney sections (10×) 24 h after IR. In contrast to the normal kidney histology from a sham rat, IR in fed rats produced tubular epithelium necrosis (asterisks) with some detached necrotic epithelial cells in the tubular lumen (arrowhead), while IR in fasted rats induced less tubular damage. (E) Western blot analysis of kidney injury molecule 1 (KIM-1); quantitative data (left) and images (right). “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05.
(leak respiration), was measured by high-resolution respirometry (oxygraph O2k, OROBOROS Instruments, Innsbruck, Austria) using 10 mM glutamate plus 2 mM malate as substrates. The changes in mitochondrial membrane potential were measured using the fluorescent dye safranin O (2 μM) as a probe in an O2k-Fluorometer (OROBOROS Instruments) [16]. The mitonuclear protein stoichiometric imbalance is presented during stressful situations [17]. In our model of IR injury, this potential imbalance was evaluated by measuring by Western blot, in isolated mitochondria, the content of the following oxidative phosphorylation proteins: nuclear DNA-encoded UQCRC2 and mitochondrial DNA-encoded MTCO1 and expressing the results as the UQCRC2/MTCO1 ratio. In addition, the content of mitochondrial chaperone HSP60 was also measured by Western blot.
magnification, comparing fed and fasted animals. To determine the extension of fibrosis, the fibrous collagen tissue deposited in the cortical areas was measured in square microns using Masson stained slides and automated image analyzer (QWin Leica, Milton Keynes, UK). The percentage of interstitial kidney fibrosis in each rat was determined comparing fed and fasted animals. For transmission electron microscopy, from the same fixed tissues, small cubic fragments (1 mm3) were obtained from the kidney cortex, that were postfixed in 2% osmium tetroxide, dehydrated, embedded in epoxy resin, ultrasectioned into 70–90 nm width, contrasted with lead and uranium salts and observed with a transmission electron microscope (Tecnai Spiriti BioTWIN, FEI, Hillsboro, OR, USA).
2.8. Histology and transmission electron microscopy
Deparaffinized tissue sections were blocked and incubated with proper dilutions of primary rabbit antibodies against 3-NT (1:200), or Nrf2 (1:200). After several washings, tissue sections were incubated with HRP-labeled secondary antibodies, which were revealed with diaminobenzidine and counter-stained with hematoxylin.
2.9. Immunohistochemistry
Sagittal sections from the kidneys were fixed by immersion in a mixed solution of 4% paraformaldehyde and 1.5% glutaraldehyde. A thin tissue slide (1 mm width) was dehydrated and embedded in paraffin, sectioned at 5 μm and stained with hematoxylin/eosin (H&E) (samples of 24-h study) and Masson trichrome (samples of 4-weeks study). For the morphometric study, the number of proximal convoluted tubules with epithelial acidophilic necrosis were counted and their percentage was determined in five random fields at 200×
2.10. Western blot analyses Tissues were lysed in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 mM 62
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Fig. 3. Fasting protects against oxidative stress induced by renal ischemiareperfusion (IR) injury. (A) Kidney protein carbonyls. (B) Assessment of 4hydroxynonenal (4-HNE) levels in kidney lysates by Western blotting. (C) Representative kidney sections (10×) immunostained for 3-nitrotyrosine (3NT). Strong immunostained tubular epithelial cells were observed in fed rats subjected to IR, while slight positive and negative immunostained cells were seen in fasted-IR and sham-operated rats, respectively. Enzymatic activities of (D) glutathione S-transferase (GST), (E) superoxide dismutase (SOD), and (F) glutathione peroxidase (GPx) in kidney homogenates after IR injury. “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05. a p < 0.05 vs Sham, bp < 0.05 vs IR.
expression were calculated by the 2−ΔΔCt method [18]. The following primers were used: Nrf2 (accession number: NM_031789): forward 5′-CACATCCAGACAGACACCAGT-3′, reverse 5′-CTACAAATGGGAATG TCTCTGC-3’; HO-1 (accession number: 012580): forward 5′-ACAGGG TGACAGAAGAGGCTAA-3′, reverse 5′-CTGTGAGGGACTCTGGTCTTTG3’; NQO-1 (accession number: NM_017000): forward 5′-CAGCGGCTCC ATGTACT-3′, reverse 5′-GACCTGGAAGCCACAGAAG-3’; β-actin (accession number: NM_031144.3): forward 5′- CTAAGGCCAACCGTGAA AAGA-3’; reverse 5′-ACAACACAGCCTGGATGGCTA-3’. β-actin was used as the housekeeping gene.
EGTA, 5 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with the protease inhibitor cocktail by using a Potter-Elvehjem tissue homogenizer and then centrifuged at 15,000 g for 10 min at 4 °C. Proteins (20 μg) were separated on SDS-polyacrylamide gel electrophoresis (PAGE) and then were electrotransferred to nitrocellulose membrane. Incubation was done overnight at 4 °C with primary antibodies against KIM-1 (1:1500), 4HNE (1:3000), Nrf2 (1:1000), tubulin (1:5000), UQCRC2/MTCO1 (1:2000), HSP60 (1:2000), and α-SMA (1:2000). Signal was detected using the Odyssey Sa imaging System (LI-COR Biosciences) and the densitometry of bands was performed with Image Studio™ Lite (Ver. 5.2, LI-COR Biosciences). Tubulin was used as loading control.
2.12. Statistical analysis Data are given as means ± SEM. Comparisons between groups were made by one-way ANOVA followed by Tukey's test using the software GraphPad Prism 5 (San Diego, CA, USA). The level of significance was set at P < 0.05.
2.11. RNA extraction and quantitative real-time PCR Total RNA was extracted using the RNeasy Kit, according to the recommendations of the manufacturer. Quality and quantity of RNA were evaluated through spectrophotometry (260/280) and on agarose gels. Reverse transcription of the mRNA was performed using 100 ng RNA, oligo-dT and the Omniscript kit. Real-time PCR was performed using Quantitect SYBR Green Mastermix kit. The samples were analyzed in duplicate, and β-actin was used as housekeeping gene. The Ct values were determined by 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and the fold changes of gene
3. Results 3.1. Protection against IR injury by fasting It was first confirmed that fasting is protective against renal IR injury. IR induced injury was characterized by increased plasma levels of 63
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Fig. 4. Fasting does not activate NF-E2-related factor 2 (Nrf2) in the kidney. (A) Total Nrf2 protein assessed in the kidney 24 h after ischemia-reperfusion (IR) injury. (B) Representative kidney sections (60×) immunostained for Nrf2. In sham-operated rats, some tubular epithelial cells showed Nrf2-positive nuclei (arrowheads), while in both fed and fasted rats subjected to IR only there was a faint cytoplasmic immunostaining. (C) Nrf2 protein assessed by Western blotting in liver and kidney lysates of rats fasted 1–3 days. “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05 vs 0 (control).
3.2. Fasting reduces IR-induced oxidative stress
Table 1 RNA expression of Nrf2 and its target genes.
Nrf2 HO-1 NQO-1
Sham
IR
IR + Fasting
1.0 1.0 1.0
2.00 ± 0.98 4.78 ± 1.13 1.26 ± 0.31
0.47 ± 0.15 3.03 ± 1.63 1.96 ± 1.21
Reperfusion following ischemia increases oxidative damage [8,9] and fasting has been shown to activate antioxidant defenses in the kidney [5,19], suggesting that this intervention may be protective against IR-induced oxidative stress. IR enhanced renal levels of protein carbonyls and 4-HNE and the abundance of 3-NTand decreased activity of GST, SOD and GPx (Fig. 3). As expected, fasting reduced the levels of the oxidative stress markers protein carbonyls and 4-HNE as well as the abundance of 3-NT (Fig. 3A and B), while increasing the activity of the antioxidant enzymes GST, SOD, and GPx in the injured kidney (Fig. 3C–E). Strikingly, we found that Nrf2, a master regulatory transcription factor of cellular antioxidant response closely associated with DR benefits [20], did not appear to be involved in fasting-mediated protection against IR injury, as fasting did not modify either total Nrf2 protein level (Fig. 4A), nor Nrf2 nuclear localization in the injured kidney. Occasional Nrf2-immunostained nuclei were observed in some proximal tubule epithelial cells from sham group, while faint Nrf2-immunostained cytoplasm, without positive nuclei, were observed in fasted and fed rats (Fig. 4B). The changes in mRNA levels of Nrf2 and
Nrf2, NF-E2-related factor 2; HO-1, heme oxygenase-1; NQO-1, NADPH quinone oxidoreductase 1; IR, Ischemia-reperfusion. Data are means ± SEM, n = 3.
BUN and creatinine and increased urinary protein excretion and expression of renal KIM-1 levels (Fig. 2). Three days of preoperative fasting reduced kidney functional decline, as shown by plasma levels of BUN and creatinine and urinary excretion of total protein 24 h after IR (Fig. 2A–C). Consistent with this, histological analysis of the kidney stained with H&E showed significantly more proximal convoluted tubules with epithelial acidophilic necrosis and detached necrotic cells in fed (28.20 ± 4.32%) than in fasted (10.31 ± 2.74%) rats (n = 3, p < 0.05) (Fig. 2D). Fasting also reduced KIM-1 protein expression (Fig. 2E), a sensitive marker of tubular damage. 64
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Fig. 5. Fasting alleviates mitochondrial dysfunction in renal ischemia-reperfusion (IR) injury. Measurement of oxygen consumption rate (OCR) during (A) state 3 and (B) state 4° respiration, and (C) changes in mitochondrial membrane potential (MMP) in isolated kidney mitochondria 24 h after IR. (D) Representative transmission electron microscopy (TEM) micrographs of mitochondria from cortical proximal tubular epithelium. Irregular-shaped mitochondria with dilated cristae are observed in fed rats after IR, while in fasted rats mitochondrial morphology was preserved. (E) Mitonuclear protein imbalance revealed by Western blot analysis of nuclear DNA (nDNA)-encoded cytochrome b-c1 complex subunit 2 (UQCRC2) and mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit 1 (MTCO1) oxidative phosphorylation proteins and heat shock protein 60 (HSP60). “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05.
Fig. 6. Fasting restrains fibrosis progression in renal ischemia-reperfusion (IR) injury. Rats were either fed ad libitum or fasted for 3 days before IR injury. (A) Alpha smooth muscle actin (α-SMA) protein assessed by Western blotting in the kidney 4 weeks after IR injury. (B) Representative Masson trichrome-stained kidney sections (10×) 4 weeks after IR. Fibrous collagen deposition (blue staining) was observed in fed rats subjected to IR, while fasted-IR animals showed scarce fibrous tissue that is similar to the sham control rats. “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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reduces cell death, inflammation, and fibrosis in murine models of renal IR injury. However, no effective strategies exist to protect against ischemic AKI in the clinic. Nutritional interventions such as DR and fasting have emerged as alternatives to enhance rodent intracellular defenses against IR injury [3], which might have important implications for AKI management and delay CKD progression. First, in the current work we provide definitive evidence that fasting reduces oxidative stress in ischemic AKI. However, in stark contrast with transcriptomic analyses showing activation of Nrf2 in the kidney upon fasting and DR [5,19], our results on the amount and intracellular distribution of Nrf2 protein (Fig. 3) indicate that this transcription factor is not involved in fasting-mediated protection against IR-induced oxidative stress; at least in the kidney, as fasting seems to specifically activate Nrf2 in the liver [25]. Also, we found that fasting, as previously suggested [26], and similar to DR in other tissues [27,28], operates through a mitochondrial pathway to protect against IR injury. Specifically, we found that fasting ameliorates disturbances in oxygen consumption, mitochondrial membrane potential, mitochondrial structure and mitonuclear protein balance. Finally, our data show that fasting might have important implications in limiting AKI to CKD transition by reducing fibrosis in the injured kidney. The mechanisms through which fasting protects against oxidative stress, mitochondrial dysfunction, and fibrosis are still unknown. For example, if Nrf2 does not mediate fasting-induced antioxidant protection in the ischemic kidney, do other transcriptional regulators such as peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1α) and Forkhead box O (FOXO)3 are involved? Also, since mitochondrial biogenesis [29], dynamics [30], and turnover [31] are tightly linked to nutrient-deprived conditions, it would be interesting to explore whether these processes involved in mitochondrial physiology contribute to beneficial effects of fasting. Finally, because oxidative stress and mitochondrial dysfunction are largely involved in fibrosis development, an issue that requires further investigation is whether fasting delays kidney fibrosis secondary to reducing both oxidative stress and mitochondrial dysfunction. Overall, our data reveal that fasting limits oxidative stress and mitochondrial dysfunction in early renal IR injury, but also has a longterm protective effect against fibrosis (Fig. 7), thus emphasizing the use of fasting as a non-pharmacological, preventive option for clinical consideration to improve the outcome of ischemic AKI.
Fig. 7. Protective effect of fasting against ischemia-reperfusion (IR) injury. Fasting limits oxidative stress and mitochondrial dysfunction in acute kidney injury (AKI), likely contributing to the long-lasting protection against fibrosis development and hence chronic kidney disease (CKD). 4-HNE, 4-hydroxynonenal; 3-NT, 3-nitrotyrosine; GST, glutathione S-transferase; SOD, superoxide dismutase; GPx, glutathione peroxidase; OCR, oxygen consumption rate; MMP, mitochondrial membrane potential; α-SMA, alpha smooth muscle actin.
those of the target genes HO-1 and NQO1 were not significant (Table 1). Furthermore, in the additional group of rats, we identified that Nrf2 protein was increased in the liver and decreased in the kidney of fasted rats (Fig. 4C). 3.3. Fasting reduces IR-induced mitochondrial dysfunction The impact of fasting was next evaluated on mitochondrial function and structure in IR injury using high-resolution respirometry and transmission electron microscopy, respectively. Isolated kidney mitochondria from fasted rats subjected to IR, displayed a significant increase in oxygen consumption rate during both ADP-stimulated (Fig. 5A) and leak (Fig. 5B) respiration, compared to fed rats. Also, fasting preserved mitochondrial membrane potential (Fig. 5C). Fed rats showed irregular mitochondrial shape with cristae dilatation and effacement in epithelial tubular cells, while the mitochondrial morphology in fasted animals was well preserved (Fig. 5D). In exploring mitonuclear protein imbalance, it was found that IR injury indeed increased the UQCRC2/MTCO1 ratio and that previous fasting prevented it (Fig. 5E). The data indicate that fasting corrected the mitonuclear imbalance induced by IR injury. This correlates with a significantly lower relative expression of HSP60 (Fig. 5E) in fasted (1.78 ± 0.21-fold induction vs. sham) than in fed (2.87 ± 0.24-fold induction vs. sham) rats (n = 3, p < 0.05). In other words, fasting prevents the IR-induced increase in HSP60.
Conflicts of interest None. Acknowledgements This work was funded by Consejo Nacional de Ciencia y Tecnologia (CONACyT, Mexico) (Grant ID 220646), Programa de Apoyo a Proyectos de Investigacion e Innovacion Tecnologica (PAPIIT, Mexico) (Grant ID IN201316), Programa de Apoyo a la Investigacion y Posgrado (PAIP, Mexico) (Grant ID 5000–9105) & by Fondos del Gasto Directo autorizado a la Subdirección de Investigación básica. Instituto Nacional de Cardiología Ignacio Chávez. We thank Dr. Ismael Torres and Dr. Enrique Pinzón for the technical support with experimental animals. P.R.M. is a doctoral student from Programa de Doctorado en Ciencias Bioquímicas, Universidad Nacional Autónoma de México (UNAM) and receives a fellowship from CONACyT.
3.4. Fasting reduces IR-induced fibrosis Finally, we evaluated the long-term effect of fasting on kidney fibrosis induced by IR injury. To this end, we analyzed α-SMA protein expression by Western blotting and collagen deposition by Masson staining in the kidney 4 weeks post-IR. We found that fasting reduced αSMA (Fig. 6A) and fibrosis in the injured kidney areas (5.16 ± 2.18 vs. 14.53 ± 5.08% in fed rats, n = 3, p < 0.05, Fig. 6B).
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4. Discussion
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Loss of both, mitochondrial homeostasis and redox balance, plays a key role in the pathophysiology of ischemic AKI. For example, targeting mitochondrial function [21,22] or treatment with antioxidants [23,24] 66
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Original article
Fasting reduces oxidative stress, mitochondrial dysfunction and fibrosis induced by renal ischemia-reperfusion injury
T
Pedro Rojas-Moralesa, Juan Carlos León-Contrerasb, Omar Emiliano Aparicio-Trejoa, Jazmin Gabriela Reyes-Ocampoa, Omar Noel Medina-Camposa, Angélica Saraí Jiménez-Osorioa, Susana González-Reyesa, Brenda Marquina-Castillob, Rogelio Hernández-Pandob, Diana Barrera-Oviedoc, Laura Gabriela Sánchez-Lozadad, José Pedraza-Chaverria, Edilia Tapiad,∗ a
Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico Departamento de Patología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Ciudad de México 14080, Mexico Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de México 04510, Mexico d Laboratorio de Fisiopatología Renal, Departamento de Nefrología, Instituto Nacional de Cardiología Ignacio Chávez, Ciudad de México 14080, Mexico b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Fasting Oxidative stress Mitochondrial dysfunction Fibrosis Ischemia-reperfusion injury Acute kidney injury Chronic kidney disease
Food deprivation protects against ischemia-reperfusion (IR) injury through unknown mechanisms. In an experimental rat model of acute IR injury, we found that preoperative fasting for 3 days protects rats from tubular damage and renal functional decline by increasing antioxidant protection independently of the NF-E2-related factor 2 (Nrf2), and by maintaining mitochondrial morphology and function. In addition, further analysis revealed that fasting protects against tubulointerstitial fibrosis. In summary, our results point out to fasting as a robust nutritional intervention to limit oxidative stress and mitochondrial dysfunction in early acute kidney injury and also to promote long-term protection against fibrosis.
1. Introduction Dietary restriction (DR) and fasting are nutritional interventions strongly associated with longevity and acute stress resistance in a disparate range of model organisms [1,2]. For example, both DR and fasting regimens delay age-related chronic diseases and also enhance intracellular defense against chemotherapy and traumatic injury in multiple tissues [2–4]. However, the underlying molecular mechanisms remain largely unknown. Early studies have shown that preoperative fasting protects mice against renal ischemia-reperfusion (IR) injury [5,6], a stressful situation that notably impairs kidney structure and function, often leading to chronic kidney disease (CKD) and high mortality in the clinic [7]. As in the seminal paper by Mitchell et al. [5] was reported that fasting increases the messenger ribonucleic acid (mRNA) of certain antioxidant proteins in IR injury, this work was rapidly embraced by the scientific community as evidence of the protective effect of fasting against IRinduced oxidative stress [4]. However, to our knowledge, no experimental evidence exists to prove this assumption. Given that oxidative stress and also mitochondrial dysfunction are the major contributing factors in the pathophysiology of IR injury
∗
[8–10], we investigated in the current work whether the protective effect of fasting is associated with maintenance of redox balance, mitochondrial function and morphology in a rat model of ischemic acute kidney injury (AKI). Additionally, we explored whether fasting confers long-lasting protection against kidney fibrosis, a maladaptive repair process after IR injury driving CKD progression [11]. 2. Materials and methods 2.1. Reagents The following reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA): Adenosine 5′-diphosphate sodium salt (ADP), antimycin A, horseradish peroxidase (HRP), bovine serum albumin (BSA) fatty acid free (A6003), BSA (A4503) 1-chloro-2,4 dinitrochlorobenzene (CDNB), carbonyl cyanide m-chlorophenylhydrazone (CCCP), sodium succinate dibasic, sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic (NaH2PO4), sodium glutamate, sodium malate, K-lactobionate, manganese (II) chloride (MgCl2) tetrahydrate, rotenone, ethylenediaminetetraacetic acid (EDTA), protease inhibitor cocktail, ethylene glycol-bis(2-aminoethylether)-
Corresponding author. E-mail address: [email protected] (E. Tapia).
https://doi.org/10.1016/j.freeradbiomed.2019.02.018 Received 23 November 2018; Received in revised form 1 February 2019; Accepted 16 February 2019 Available online 25 February 2019 0891-5849/ © 2019 Elsevier Inc. All rights reserved.
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N,N,N′,N′-tetraacetic acid (EGTA), glutamate, glutaraldehyde, glutathione (GSH), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), malate, D-mannitol, sodium chloride (NaCl), nicotinamide adenine dinucleotide phosphate (NADPH), sodium fluoride (NaF), sodium orthovanadate (Na3VO4), nitroblue tetrazolium (NBT), Nonidet P40 (NP-40), paraformaldehyde, phenylmethylsulfonyl fluoride (PMSF), safranin O, sodium deoxycholate, sodium dodecyl sulfate (SDS), sucrose, taurine, Tris-HCl, xanthine, xanthine oxidase, dinitrophenylhydrazine, streptomycin sulfate, guanidine hydrochloride and primary antibodies against tubulin (#T9026). Ethanol, hydrochloric acid, ethyl acetate, and trichloroacetic acid were from JT Baker (Xalostoc, Mexico). Antibodies against kidney injury molecule 1 (KIM-1) were from R&D systems (Minneapolis, MN, USA). Antibodies against 3-nitrotyrosine (3-NT, #9691) were from Cell Signaling (Danvers, MA, USA). Primary antibodies against 4-hydroxynoneal (4-HNE, #AB5605) were from Merck Millipore (Burlington, MA, USA). Primary antibodies against NF-E2-related factor 2 (Nrf2, #sc-722) were from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies against cytochrome c oxidase subunit 1 (MTCO1) [1D6E1A8] (#ab14705), cytochrome b-c1 complex subunit 2 (UQCRC2) [13G12AF12BB11] (#ab14745), and heat shock protein 60 (HSP60, #ab31115) were from Abcam (Cambridge, MA, USA). Primary antibodies against alpha smooth muscle actin (α-SMA, #GTX100034) were from GeneTex (Irvine, CA, USA). The IRDye® secondary antibodies were acquired from LI-COR Biosciences (Lincoln, NE, USA). Commercial kits to measure blood urea nitrogen (BUN) and creatinine levels in plasma were obtained from SpinReact (Girona, Spain). The Bio-Rad protein assay dye reagent concentrate to measure total protein (Bradford assay) was from Bio Rad Laboratories Inc. (Hercules, CA, USA). The following kits RNeasy, Omniscript reverse transcriptase (RT) and QuantiTect SYBR Green polymerase chain reaction (PCR) were form Qiagen (Germantown, MD, USA).
Fig. 1. Experimental designs. (A) To investigate the protective effect of fasting, rats were either fed ad libitum or fasted for 3 days before ischemia-reperfusion (IR) injury. 24 h after IR, rats were euthanized to analyze renal damage, oxidative stress markers and mitochondrial function. At 28 days post IR injury, rats were euthanized to analyze fibrosis development. (B) To analyze Nrf2 protein expression in the liver and kidney rats were fasted for 1–3 days without IR injury.
deposition (Fig. 1). 2.4. Renal function analysis Creatinine and BUN levels were assessed in plasma by commercial kits according to manufacturer's instructions. For proteinuria determination, rats were housed individually in metabolic cages and urine was collected over a 24-h period. Urinary protein excretion was measured by the Bradford assay [14].
2.2. Experimental animals
2.5. Protein carbonyls
Male Wistar rats (8–9 weeks old, n = 5–6 per group) weighing 240–260 g were housed in standard laboratory conditions. Rats were divided in three groups: (a) Sham, (b) fasted (beginning at 10 a.m.) for 3 days before IR injury, (c) fed before IR injury. An additional group of rats was fasted during 1, 2 or 3 days and euthanized to obtain kidneys and livers to measure Nrf2 levels. Experiments were approved by the Animal care committee (CICUAL) of the National Institute of Cardiology “Ignacio Chávez” (#18–1050).
For protein carbonylation determination, tissue extracts were derivatized with 10 mM 2,4-dinitrophenylhydrazine for 1 h and then proteins were precipitated with 20% trichloroacetic acid. After washing for several times with an ethanol-ethyl acetate mixture (1:1 v/v), proteins were solubilized in 6 M guanidine hydrochloride and the absorbance was recorded at 370 nm [15].
2.3. IR injury model
Enzyme activities were assessed in kidney lysates as previously described [16]. Briefly, GST activity was assessed by following the formation of the GSH-CDNB adduct at 340 nm. SOD activity was measured at 560 nm as the inhibition of NBT reduction in a superoxidegenerating system by xanthine-xanthine oxidase. GPx activity was measured by following NADPH disappearance at 340 nm in a spectrophotometer in a GSH-regenerating system [16].
2.6. Enzyme assays
An established rat model of AKI was used, induced by unilateral IR injury plus contralateral nephrectomy, as previously described [12,13]. Briefly, under sodium pentobarbital anesthesia (60 mg/kg, i.p.), the right kidney was excised and ischemia was done in the left kidney by clamping the renal artery for 30 min, using non-traumatic vascular clips. For sham surgery, the kidneys were exposed but the renal artery was not clamped. After surgery, rats were fed ad libitum and sacrificed 24 h or 4 weeks later to harvest blood and the remaining kidney for analysis (Fig. 1). The following parameters were measured in the group of 24 h: markers of biochemical and structural renal damage (plasma levels of creatinine and BUN, renal KIM-1 levels and kidney histological studies by light microscopy), activity of the antioxidant enzymes glutathione S-transferase (GST), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in kidney lysates, markers of oxidant stress (protein carbonyls, 4-HNE, and 3-NT), renal Nrf2 levels, mRNA levels of Nrf2, heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase-1 (NQO-1), markers of mitochondrial function (oxygen consumption, mitochondrial membrane potential, mitochondrial ultrastructure and mitonuclear protein imbalance). Renal fibrosis was measured in the group of 4 weeks by measuring α-SMA levels and fibrous collagen
2.7. Mitochondrial function To evaluate mitochondrial function, kidney mitochondria were isolated by differential centrifugation in cold (4 °C) isolation buffer (225 mM D-mannitol, 75 mM sucrose, 1 mM EDTA, 5 mM HEPES, 0.1% BSA, pH 7.4) as previously described [16]. The final mitochondria pellet was resuspended in 100 μL of BSA-free isolation buffer and the mitochondrial total protein was measured by the Lowry method. Isolated mitochondria (200 μg of total protein) were loaded into the chamber that contained 2 ml of MiR05 respiration buffer: 0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose and 1 g/L BSA essentially fatty acid free. Mitochondrial oxygen consumption rate during state 3 (ADP-stimulated, 2.5 mM) and state 4°, induced by 2.5 μM oligomycin 61
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Fig. 2. Protective effect of fasting against biochemical and structural renal damage induced by renal ischemia-reperfusion (IR). Rats were either fed ad libitum or fasted for 3 days before IR injury. Plasma levels of (A) blood urea nitrogen (BUN) and (B) creatinine, and (C) urinary protein excretion assessed 24 h after IR. (D) Representative hematoxylin and eosin (H&E)-stained kidney sections (10×) 24 h after IR. In contrast to the normal kidney histology from a sham rat, IR in fed rats produced tubular epithelium necrosis (asterisks) with some detached necrotic epithelial cells in the tubular lumen (arrowhead), while IR in fasted rats induced less tubular damage. (E) Western blot analysis of kidney injury molecule 1 (KIM-1); quantitative data (left) and images (right). “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05.
(leak respiration), was measured by high-resolution respirometry (oxygraph O2k, OROBOROS Instruments, Innsbruck, Austria) using 10 mM glutamate plus 2 mM malate as substrates. The changes in mitochondrial membrane potential were measured using the fluorescent dye safranin O (2 μM) as a probe in an O2k-Fluorometer (OROBOROS Instruments) [16]. The mitonuclear protein stoichiometric imbalance is presented during stressful situations [17]. In our model of IR injury, this potential imbalance was evaluated by measuring by Western blot, in isolated mitochondria, the content of the following oxidative phosphorylation proteins: nuclear DNA-encoded UQCRC2 and mitochondrial DNA-encoded MTCO1 and expressing the results as the UQCRC2/MTCO1 ratio. In addition, the content of mitochondrial chaperone HSP60 was also measured by Western blot.
magnification, comparing fed and fasted animals. To determine the extension of fibrosis, the fibrous collagen tissue deposited in the cortical areas was measured in square microns using Masson stained slides and automated image analyzer (QWin Leica, Milton Keynes, UK). The percentage of interstitial kidney fibrosis in each rat was determined comparing fed and fasted animals. For transmission electron microscopy, from the same fixed tissues, small cubic fragments (1 mm3) were obtained from the kidney cortex, that were postfixed in 2% osmium tetroxide, dehydrated, embedded in epoxy resin, ultrasectioned into 70–90 nm width, contrasted with lead and uranium salts and observed with a transmission electron microscope (Tecnai Spiriti BioTWIN, FEI, Hillsboro, OR, USA).
2.8. Histology and transmission electron microscopy
Deparaffinized tissue sections were blocked and incubated with proper dilutions of primary rabbit antibodies against 3-NT (1:200), or Nrf2 (1:200). After several washings, tissue sections were incubated with HRP-labeled secondary antibodies, which were revealed with diaminobenzidine and counter-stained with hematoxylin.
2.9. Immunohistochemistry
Sagittal sections from the kidneys were fixed by immersion in a mixed solution of 4% paraformaldehyde and 1.5% glutaraldehyde. A thin tissue slide (1 mm width) was dehydrated and embedded in paraffin, sectioned at 5 μm and stained with hematoxylin/eosin (H&E) (samples of 24-h study) and Masson trichrome (samples of 4-weeks study). For the morphometric study, the number of proximal convoluted tubules with epithelial acidophilic necrosis were counted and their percentage was determined in five random fields at 200×
2.10. Western blot analyses Tissues were lysed in cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1 mM 62
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Fig. 3. Fasting protects against oxidative stress induced by renal ischemiareperfusion (IR) injury. (A) Kidney protein carbonyls. (B) Assessment of 4hydroxynonenal (4-HNE) levels in kidney lysates by Western blotting. (C) Representative kidney sections (10×) immunostained for 3-nitrotyrosine (3NT). Strong immunostained tubular epithelial cells were observed in fed rats subjected to IR, while slight positive and negative immunostained cells were seen in fasted-IR and sham-operated rats, respectively. Enzymatic activities of (D) glutathione S-transferase (GST), (E) superoxide dismutase (SOD), and (F) glutathione peroxidase (GPx) in kidney homogenates after IR injury. “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05. a p < 0.05 vs Sham, bp < 0.05 vs IR.
expression were calculated by the 2−ΔΔCt method [18]. The following primers were used: Nrf2 (accession number: NM_031789): forward 5′-CACATCCAGACAGACACCAGT-3′, reverse 5′-CTACAAATGGGAATG TCTCTGC-3’; HO-1 (accession number: 012580): forward 5′-ACAGGG TGACAGAAGAGGCTAA-3′, reverse 5′-CTGTGAGGGACTCTGGTCTTTG3’; NQO-1 (accession number: NM_017000): forward 5′-CAGCGGCTCC ATGTACT-3′, reverse 5′-GACCTGGAAGCCACAGAAG-3’; β-actin (accession number: NM_031144.3): forward 5′- CTAAGGCCAACCGTGAA AAGA-3’; reverse 5′-ACAACACAGCCTGGATGGCTA-3’. β-actin was used as the housekeeping gene.
EGTA, 5 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with the protease inhibitor cocktail by using a Potter-Elvehjem tissue homogenizer and then centrifuged at 15,000 g for 10 min at 4 °C. Proteins (20 μg) were separated on SDS-polyacrylamide gel electrophoresis (PAGE) and then were electrotransferred to nitrocellulose membrane. Incubation was done overnight at 4 °C with primary antibodies against KIM-1 (1:1500), 4HNE (1:3000), Nrf2 (1:1000), tubulin (1:5000), UQCRC2/MTCO1 (1:2000), HSP60 (1:2000), and α-SMA (1:2000). Signal was detected using the Odyssey Sa imaging System (LI-COR Biosciences) and the densitometry of bands was performed with Image Studio™ Lite (Ver. 5.2, LI-COR Biosciences). Tubulin was used as loading control.
2.12. Statistical analysis Data are given as means ± SEM. Comparisons between groups were made by one-way ANOVA followed by Tukey's test using the software GraphPad Prism 5 (San Diego, CA, USA). The level of significance was set at P < 0.05.
2.11. RNA extraction and quantitative real-time PCR Total RNA was extracted using the RNeasy Kit, according to the recommendations of the manufacturer. Quality and quantity of RNA were evaluated through spectrophotometry (260/280) and on agarose gels. Reverse transcription of the mRNA was performed using 100 ng RNA, oligo-dT and the Omniscript kit. Real-time PCR was performed using Quantitect SYBR Green Mastermix kit. The samples were analyzed in duplicate, and β-actin was used as housekeeping gene. The Ct values were determined by 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and the fold changes of gene
3. Results 3.1. Protection against IR injury by fasting It was first confirmed that fasting is protective against renal IR injury. IR induced injury was characterized by increased plasma levels of 63
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Fig. 4. Fasting does not activate NF-E2-related factor 2 (Nrf2) in the kidney. (A) Total Nrf2 protein assessed in the kidney 24 h after ischemia-reperfusion (IR) injury. (B) Representative kidney sections (60×) immunostained for Nrf2. In sham-operated rats, some tubular epithelial cells showed Nrf2-positive nuclei (arrowheads), while in both fed and fasted rats subjected to IR only there was a faint cytoplasmic immunostaining. (C) Nrf2 protein assessed by Western blotting in liver and kidney lysates of rats fasted 1–3 days. “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05 vs 0 (control).
3.2. Fasting reduces IR-induced oxidative stress
Table 1 RNA expression of Nrf2 and its target genes.
Nrf2 HO-1 NQO-1
Sham
IR
IR + Fasting
1.0 1.0 1.0
2.00 ± 0.98 4.78 ± 1.13 1.26 ± 0.31
0.47 ± 0.15 3.03 ± 1.63 1.96 ± 1.21
Reperfusion following ischemia increases oxidative damage [8,9] and fasting has been shown to activate antioxidant defenses in the kidney [5,19], suggesting that this intervention may be protective against IR-induced oxidative stress. IR enhanced renal levels of protein carbonyls and 4-HNE and the abundance of 3-NTand decreased activity of GST, SOD and GPx (Fig. 3). As expected, fasting reduced the levels of the oxidative stress markers protein carbonyls and 4-HNE as well as the abundance of 3-NT (Fig. 3A and B), while increasing the activity of the antioxidant enzymes GST, SOD, and GPx in the injured kidney (Fig. 3C–E). Strikingly, we found that Nrf2, a master regulatory transcription factor of cellular antioxidant response closely associated with DR benefits [20], did not appear to be involved in fasting-mediated protection against IR injury, as fasting did not modify either total Nrf2 protein level (Fig. 4A), nor Nrf2 nuclear localization in the injured kidney. Occasional Nrf2-immunostained nuclei were observed in some proximal tubule epithelial cells from sham group, while faint Nrf2-immunostained cytoplasm, without positive nuclei, were observed in fasted and fed rats (Fig. 4B). The changes in mRNA levels of Nrf2 and
Nrf2, NF-E2-related factor 2; HO-1, heme oxygenase-1; NQO-1, NADPH quinone oxidoreductase 1; IR, Ischemia-reperfusion. Data are means ± SEM, n = 3.
BUN and creatinine and increased urinary protein excretion and expression of renal KIM-1 levels (Fig. 2). Three days of preoperative fasting reduced kidney functional decline, as shown by plasma levels of BUN and creatinine and urinary excretion of total protein 24 h after IR (Fig. 2A–C). Consistent with this, histological analysis of the kidney stained with H&E showed significantly more proximal convoluted tubules with epithelial acidophilic necrosis and detached necrotic cells in fed (28.20 ± 4.32%) than in fasted (10.31 ± 2.74%) rats (n = 3, p < 0.05) (Fig. 2D). Fasting also reduced KIM-1 protein expression (Fig. 2E), a sensitive marker of tubular damage. 64
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Fig. 5. Fasting alleviates mitochondrial dysfunction in renal ischemia-reperfusion (IR) injury. Measurement of oxygen consumption rate (OCR) during (A) state 3 and (B) state 4° respiration, and (C) changes in mitochondrial membrane potential (MMP) in isolated kidney mitochondria 24 h after IR. (D) Representative transmission electron microscopy (TEM) micrographs of mitochondria from cortical proximal tubular epithelium. Irregular-shaped mitochondria with dilated cristae are observed in fed rats after IR, while in fasted rats mitochondrial morphology was preserved. (E) Mitonuclear protein imbalance revealed by Western blot analysis of nuclear DNA (nDNA)-encoded cytochrome b-c1 complex subunit 2 (UQCRC2) and mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit 1 (MTCO1) oxidative phosphorylation proteins and heat shock protein 60 (HSP60). “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05.
Fig. 6. Fasting restrains fibrosis progression in renal ischemia-reperfusion (IR) injury. Rats were either fed ad libitum or fasted for 3 days before IR injury. (A) Alpha smooth muscle actin (α-SMA) protein assessed by Western blotting in the kidney 4 weeks after IR injury. (B) Representative Masson trichrome-stained kidney sections (10×) 4 weeks after IR. Fibrous collagen deposition (blue staining) was observed in fed rats subjected to IR, while fasted-IR animals showed scarce fibrous tissue that is similar to the sham control rats. “-“ represents fed rats. Data are means ± SEM, n = 5–6, *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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reduces cell death, inflammation, and fibrosis in murine models of renal IR injury. However, no effective strategies exist to protect against ischemic AKI in the clinic. Nutritional interventions such as DR and fasting have emerged as alternatives to enhance rodent intracellular defenses against IR injury [3], which might have important implications for AKI management and delay CKD progression. First, in the current work we provide definitive evidence that fasting reduces oxidative stress in ischemic AKI. However, in stark contrast with transcriptomic analyses showing activation of Nrf2 in the kidney upon fasting and DR [5,19], our results on the amount and intracellular distribution of Nrf2 protein (Fig. 3) indicate that this transcription factor is not involved in fasting-mediated protection against IR-induced oxidative stress; at least in the kidney, as fasting seems to specifically activate Nrf2 in the liver [25]. Also, we found that fasting, as previously suggested [26], and similar to DR in other tissues [27,28], operates through a mitochondrial pathway to protect against IR injury. Specifically, we found that fasting ameliorates disturbances in oxygen consumption, mitochondrial membrane potential, mitochondrial structure and mitonuclear protein balance. Finally, our data show that fasting might have important implications in limiting AKI to CKD transition by reducing fibrosis in the injured kidney. The mechanisms through which fasting protects against oxidative stress, mitochondrial dysfunction, and fibrosis are still unknown. For example, if Nrf2 does not mediate fasting-induced antioxidant protection in the ischemic kidney, do other transcriptional regulators such as peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1α) and Forkhead box O (FOXO)3 are involved? Also, since mitochondrial biogenesis [29], dynamics [30], and turnover [31] are tightly linked to nutrient-deprived conditions, it would be interesting to explore whether these processes involved in mitochondrial physiology contribute to beneficial effects of fasting. Finally, because oxidative stress and mitochondrial dysfunction are largely involved in fibrosis development, an issue that requires further investigation is whether fasting delays kidney fibrosis secondary to reducing both oxidative stress and mitochondrial dysfunction. Overall, our data reveal that fasting limits oxidative stress and mitochondrial dysfunction in early renal IR injury, but also has a longterm protective effect against fibrosis (Fig. 7), thus emphasizing the use of fasting as a non-pharmacological, preventive option for clinical consideration to improve the outcome of ischemic AKI.
Fig. 7. Protective effect of fasting against ischemia-reperfusion (IR) injury. Fasting limits oxidative stress and mitochondrial dysfunction in acute kidney injury (AKI), likely contributing to the long-lasting protection against fibrosis development and hence chronic kidney disease (CKD). 4-HNE, 4-hydroxynonenal; 3-NT, 3-nitrotyrosine; GST, glutathione S-transferase; SOD, superoxide dismutase; GPx, glutathione peroxidase; OCR, oxygen consumption rate; MMP, mitochondrial membrane potential; α-SMA, alpha smooth muscle actin.
those of the target genes HO-1 and NQO1 were not significant (Table 1). Furthermore, in the additional group of rats, we identified that Nrf2 protein was increased in the liver and decreased in the kidney of fasted rats (Fig. 4C). 3.3. Fasting reduces IR-induced mitochondrial dysfunction The impact of fasting was next evaluated on mitochondrial function and structure in IR injury using high-resolution respirometry and transmission electron microscopy, respectively. Isolated kidney mitochondria from fasted rats subjected to IR, displayed a significant increase in oxygen consumption rate during both ADP-stimulated (Fig. 5A) and leak (Fig. 5B) respiration, compared to fed rats. Also, fasting preserved mitochondrial membrane potential (Fig. 5C). Fed rats showed irregular mitochondrial shape with cristae dilatation and effacement in epithelial tubular cells, while the mitochondrial morphology in fasted animals was well preserved (Fig. 5D). In exploring mitonuclear protein imbalance, it was found that IR injury indeed increased the UQCRC2/MTCO1 ratio and that previous fasting prevented it (Fig. 5E). The data indicate that fasting corrected the mitonuclear imbalance induced by IR injury. This correlates with a significantly lower relative expression of HSP60 (Fig. 5E) in fasted (1.78 ± 0.21-fold induction vs. sham) than in fed (2.87 ± 0.24-fold induction vs. sham) rats (n = 3, p < 0.05). In other words, fasting prevents the IR-induced increase in HSP60.
Conflicts of interest None. Acknowledgements This work was funded by Consejo Nacional de Ciencia y Tecnologia (CONACyT, Mexico) (Grant ID 220646), Programa de Apoyo a Proyectos de Investigacion e Innovacion Tecnologica (PAPIIT, Mexico) (Grant ID IN201316), Programa de Apoyo a la Investigacion y Posgrado (PAIP, Mexico) (Grant ID 5000–9105) & by Fondos del Gasto Directo autorizado a la Subdirección de Investigación básica. Instituto Nacional de Cardiología Ignacio Chávez. We thank Dr. Ismael Torres and Dr. Enrique Pinzón for the technical support with experimental animals. P.R.M. is a doctoral student from Programa de Doctorado en Ciencias Bioquímicas, Universidad Nacional Autónoma de México (UNAM) and receives a fellowship from CONACyT.
3.4. Fasting reduces IR-induced fibrosis Finally, we evaluated the long-term effect of fasting on kidney fibrosis induced by IR injury. To this end, we analyzed α-SMA protein expression by Western blotting and collagen deposition by Masson staining in the kidney 4 weeks post-IR. We found that fasting reduced αSMA (Fig. 6A) and fibrosis in the injured kidney areas (5.16 ± 2.18 vs. 14.53 ± 5.08% in fed rats, n = 3, p < 0.05, Fig. 6B).
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