Free Radical Research
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: http://www.tandfonline.com/loi/ifra20
Kidney fibrosis is independent of the amount of ascorbic acid in mice with unilateral ureteral obstruction H. Nishida, T. Kurahashi, Y. Saito, N. Otsuki, M. Kwon, H. Ohtake, M. Yamakawa, K.-I. Yamada, S. Miyata, Y. Tomita & J. Fujii To cite this article: H. Nishida, T. Kurahashi, Y. Saito, N. Otsuki, M. Kwon, H. Ohtake, M. Yamakawa, K.-I. Yamada, S. Miyata, Y. Tomita & J. Fujii (2014) Kidney fibrosis is independent of the amount of ascorbic acid in mice with unilateral ureteral obstruction, Free Radical Research, 48:9, 1115-1124 To link to this article: http://dx.doi.org/10.3109/10715762.2014.915031
View supplementary material
Accepted online: 15 Apr 2014.Published online: 21 Jul 2014.
Submit your article to this journal
Article views: 80
View related articles
View Crossmark data
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ifra20 Download by: [Karolinska Institutet, University Library]
Date: 12 September 2015, At: 16:30
Free Radical Research, September 2014; 48(9): 1115–1124 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.915031
ORIGINAL ARTICLE
Kidney fibrosis is independent of the amount of ascorbic acid in mice with unilateral ureteral obstruction H. Nishida1,2, T. Kurahashi1, Y. Saito1, N. Otsuki1, M. Kwon1, H. Ohtake3, M. Yamakawa3, K.-I. Yamada4, S. Miyata5, Y. Tomita2 & J. Fujii1 1Department
of Biochemistry and Molecular Biology, Yamagata University School of Medicine, Yamagata, Japan, of Urology, Yamagata University School of Medicine, Yamagata, Japan, 3Department of Diagnostic Pathology, Faculty of Medicine, Yamagata University, Yamagata, Japan, 4Department of Bio-functional Science, Faculty of Pharmacological Science, Kyushu University, Fukuoka, Japan, and 5Department of Internal Medicine, Osaka Koseinenkin Hospital, Osaka, Japan
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
2Department
Abstract In response to sustained damage to a kidney, fibrosis that can be characterized as the deposition of a collagenous matrix occurs and consequently causes chronic kidney failure. Because most animals used in experiments synthesize ascorbic acid (AsA) from glucose, the roles of AsA in fibrotic kidney diseases are largely unknown. Unilateral ureteric obstruction (UUO) mimics the complex pathophysiology of chronic obstructive nephropathy and is an ideal model for the investigation of the roles of AsA in kidney failure. We examined the impact of a deficiency of Akr1a, a gene that encodes aldehyde reductase and is responsible for the production of AsA, on fibrotic damage caused by UUO in mice. Oxidatively modified DNA was elevated in wild-type and Akr1a-deficient kidneys as a result of UUO to a similar extent, and was only slightly suppressed by the administration of AsA. Even though Akrla-deficient mice could produce only about 10% of the AsA produced by wild-type mice, no difference was observed in collagen I synthesis under pathological conditions. The data implied either a low demand for AsA or the presence of another electron donor for collagen I production in the mouse kidney. Next, we attempted to elucidate the potential causes for oxidative damage in kidney cells during the fibrotic change. We found decreases in mitochondrial proteins, particularly in electron transport complexes, at the initial stage of the kidney fibrosis. The data imply that a dysfunction of the mitochondria leads to an elevation of ROS, which results in kidney fibrosis by stimulating cellular transformation to myofibroblasts. Keywords: mitochondria, kidney, oxidative stress
Introduction In response to sustained damage to a kidney, fibrosis characterized as the deposition of a collagenous matrix occurs and consequently causes chronic kidney failure. Unilateral ureteric obstruction (UUO) is an experimental model of renal injury that mimics the complex pathophysiology of chronic obstructive nephropathy [1,2]. The hydrostatic pressure induced by UUO leads to tubular cell death, interstitial inflammatory infiltration, capillary rarefaction, loss of renal parenchyma, and progressive fibrosis by activating myofibroblasts [3–5]. In this condition, oxidative stress markers such as malondialdehyde, 8-hydroxyguanine (8-OHdG), and 8-isoprostaglandin F2αl also are elevated [6,7], and mice that lack catalase are more susceptible to UUO-induced renal injury compared with wild-type mice [8]. Thus, oxidative stress is involved in the pathogenesis of UUO, and NADPH oxidase from inflammatory cells has been implicated as the potential source for reactive oxygen species (ROS) [9]. Other than catalase, antioxidative enzymes that include superoxide dismutase (SOD), glutathione peroxidase (GPX), and peroxiredoxin play roles in suppressing oxidative injury during the inflammatory process and in other
diseases. In addition to the enzymes, low molecular weight compounds such as glutathione and vitamin C (ascorbic acid; AsA) also function to maintain the redox balance of the cells by eliminating ROS [10,11]. On the other hand, AsA is an important cofactor for some oxygenases and prolyl hydroxylase and participates in a variety of physiological reactions such as collagen synthesis, adrenaline production, and steroidogenesis [12,13]. The nephroprotective effects of AsA have been documented under some disease conditions [14–16], although ambiguity remains from a clinical point of view. While most vertebrates including rodents can naturally synthesize AsA, humans cannot due to a loss of function of L-gulono-γ-lactone oxidase (Gulo) that localizes in the endoplasmic reticulum (ER) membrane and catalyzes the last step of AsA synthesis [13]. Recently our group and others have found that aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is the enzyme responsible for the production of AsA by reducing D-glucuronic acid in mice [17,18]. Aldose reductase (AKR1B), a closely related family member, also catalyzes the same reaction although its contribution to AsA synthesis is ∼15% in the mouse body [17].
Correspondence: Junichi Fujii, Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan. Tel: 81-23-628-5227. Fax: 81-23-628-5230. E-mail: [email protected] (Received date: 27 January 2014; Accepted date: 10 April 2014; Published online: 12 May 2014)
1116 H. Nishida et al. Here, we examined whether Akr1a deficiency affects collagen synthesis under the pathological conditions elicited by UUO. We also found mitochondrial dysfunction to be a likely cause for ROS production.
Experimental
Akr1a knockout (KO) mice were generated using the gene-targeting technique and were genotyped by PCR, as described in the literature [18]. After backcrosses to C57BL/6 N mice for more than 8 times, Akr1a/ (WT) and Akr1a/ (KO) littermates were obtained [19]. The WT and KO mice were weaned at 30 days of age and fed either an AsA-deficient diet (CL-2; CLEA Japan, Tokyo) or a standard diet (Picolab 5053, LabDiet, St Louis, MO) ad libitum with free access to either water or water containing 1.5 mg/ml AsA until use. All animal experiments were performed in accordance with the Declaration of Helsinki under the protocol approved by the Animal Research Committee of this institution.
Blood test Blood was collected in the presence of excess ethylenediaminetetraacetic acid (EDTA) from the tail vein at the time of ureter ligation and from the inferior vena cava at the time of kidney harvest. After centrifugation, the plasma concentrations of blood urea nitrogen (BUN) and creatinine were determined using Fuji Drychem 3500 V and Fuji Dry-chem slides (Fuji film), respectively.
(A)
50
Surgical operation and experimental design
Histological and immunohistochemical analyses Tissue sections 2 to 3 μm in thickness were rehydrated and subjected to periodic acid-Schiff (PAS) and Elastica Masson staining for the evaluation of kidney injury. Rehydrated tissue sections were also subjected to immunohistochemical analysis. Briefly, we incubated the tissue sections with 3% hydrogen peroxide solution to eliminate intrinsic peroxidase activity. The target retrieval procedure involved the immersion of tissue sections in a citrate-based buffer solution followed by heating in an autoclave. After rinsing, the sections were incubated for 10 min with 2% BSA/TBS at room temperature, followed by incubation with a rabbit anti-rat AKR1A polyclonal antibody [20] and a mouse anti-8-hydroxydeoxyguanine
WT KO KO+ASA
40 Body weight (g)
Male mice were anesthetized via the inhalation of isoflurane [19]. Ureteral obstruction was achieved on male mice at 9–10 weeks of age by ligating the left proximal ureter with 4-0 silk through a left lateral incision. The mice were euthanized on either 5 days in most experiments or 0, 1, 2, 3, 5, and 7 days in a time course experiment after surgical operation under ether anesthesia, and the kidneys were harvested for evaluation. The right-side kidneys of the mice not subjected to the operation were also harvested for control samples. The harvested kidneys were bisected just after excision. One-half of the kidneys was frozen in liquid nitrogen immediately, and then it was stored at –80°C for determination of proteins and lipid peroxidation. The other half was fixed in 10% buffered formalin followed by embedding in paraffin.
30
20
10 8
(B)
16
24
32 40 Age (weeks)
48
56
100
80 Survival rate (%)
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Animals
(8-OHdG) monoclonal antibody (MOG-100 at a concentration of 5 μg/ml; Nikken Seil, Shizuoka) overnight in a humidified chamber at 4°C. After rinsing, the sections were sequentially reacted with peroxidase-labeled goat anti-rabbit and mouse IgG polymer for 30 min. After a final rinse, specific immunolabeling was visualized by placing a chromogen, 3,3′-diaminobenzine (K1391, Dako, USA) on the tissue sections for a few minutes. The sections were then dehydrated, mounted, and examined via light microscopy.
60
40 WT
20
KO KO+AsA
0 0
10
20
30
40
50
Age (weeks)
Figure 1. Changes in the body weight and survival rates of KO mice with and without the administration of AsA. (A) Age-dependent changes in the body weight of the WT, KO, and KO AsA mice. (B) Kaplan–Meier analysis was performed for each group. Mean SEM for 10 female mice.
Mitochondrial dysfunction in renal failure
Measurement of plasma ascorbic acid
curve constructed using 0, 10, 25, 50, 75, 100 μM of AsA. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis Kidneys were homogenized in RIPA Buffer and centrifuged at 17 400 g in a microcentrifuge. The supernatant was used for protein assay via a BCA protein assay reagent (Thermo Fisher Scientific, Yokohama, Japan). The protein samples (30 or 40 μg) were subjected to 7.5–15% sodium dodecylsulfate-polyacrylamide gel electrophoresis
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
To detect the AsA in plasma, 5 μl of plasma was added to an assay solution containing 85 μl of distilled water and 10 μl of 500 μM 15-(naphthalen-1-ylamino)-7aza-3,11-dioxadispiro[5.1.5 8 .3 6 ]hexadecan-7 - oxyl (Naph-DiPy) fluorescent indicator [21]. After a 30-min reaction at room temperature, the fluorescence was measured at an excitation wavelength of 310 nm and an emission wavelength of 430 nm using a Valioskan Flash microplate reader (Thermo Fisher Scientific). The concentrations were calculated from a standard
1117
Figure 2. Expression of AKR1A in kidneys and plasma levels of AsA in WT and KO mice. (A) Expression of AKR1A and AKR1B was detected by immunoblot analysis using anti-AKR1A antibody. (B) Immunohistochemical detection of AKR1A in the kidneys of WT and KO mice. Typical data from several experiments were shown. 400. Bars 50 μm. (C) Plasma levels of AsA were measured using a fluorescent indicator. Means SD are shown.
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
1118 H. Nishida et al. (SDS-PAGE) and electroblotted onto PVDF membranes (GE-Health care). The membranes were then blocked by incubation with 3% skimmed milk in TBST (150 mM NaCl and 20 mM Tris/HCl, pH7.6, 0.1% Tween-20) for 1 h at room temperature. The membranes were then incubated with antibodies to AKR1A [22], aldose reductase (AKR1B) [20], SOD1 [23], SOD2 [23], PRDX1 [23], PRDX4 [24], glutathione peroxidase 1 (GPX1) [25], catalase (219010, Carbiochem-Merck Bioscience, Billerica, MA, USA), cytochrome c (cyt c; Pharmingen), the mouse monoclonal antibody against β-actin (sc-69879, Santa Cruz Biotechnology), or a total Oxphos rodent antibody cocktail that included five antibodies against mitochondrial electron transfer complexes (METC) (ab11013, Abcam). After washing with TBST, the membrane was incubated with a 1:20 000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz Biotechnology) for 1 h at room temperature. After washing with TBST, the immunoreactive bands were detected by measuring chemiluminescence using Immobilon Western (Millipore) on an image analyzer (ImageQuant LAS500, GE Healthcare). Each protein amount was semi-quantified using NIH image software, and the relative values are shown.
test followed by a Tukey–Kramer multiple comparison test. A p-value of less than 0.05 was considered significant.
Statistical analysis
AKR1A expression and localization in the mouse kidney
The results are expressed as the mean SD. Comparison between groups was performed using a one-way ANOVA
We examined the expression and localization of AKR1A in the kidneys of WT and KO mice. While the kidneys of
Results AsA deficiency causes weight loss and premature death in aged AKR1A-KO mice We first investigated the body weight and the lifespan of KO mice and compared them with those of WT mice and KO mice supplemented with AsA in drinking water (KO AsA). The body weight of the KO mice was the same as the mice in the other group up to ∼20 weeks, at which time it decreased (Figure 1A). The KO mice started dying around 10 weeks of age and all had died within 1 year under the AsA-deficient diet (Figure 1B). However, supplementation of AsA (1.5 mg/ml in drinking water) contributed to maintaining body weight and extended the life-span of the KO mice. This suggests that the deaths were caused mainly by AsA deficiency due to the lack of AKR1A and that the lives of these mice could not have been supported by the AsA that was produced via AKR1B function alone.
Figure 3. Histological appearance of the kidneys of WT and KO mice with and without the administration of AsA. Kidney sections of WT, KO, and KO AsA mice were stained with PAS (upper panels) or Elastica Masson (lower panels). Magnification: 100; Bars 200 μm.
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Mitochondrial dysfunction in renal failure
the WT mice showed bands at corresponding positions of AKR1A and AKR1B, no detectable AKR1A proteins were observed in the KO kidneys (Figure 2A). Immunohistochemical staining using the anti-AKR1A antibody revealed that AKR1A was highly expressed in the epithelial cells lining the Bowman’s capsules and proximal tubules, particularly on the brush border (Figure 2B). Because AKR1A is involved in AsA synthesis [17,18], we measured the plasma levels of AsA in WT mice, KO mice, and KO mice supplemented with AsA in drinking water (KO AsA) and found these levels initially to be very low in the KO mice compared with the WT mice, but these levels recovered to wild-type levels after AsA supplementation (Figure 2C). Because AsA levels in the KO mice fed a standard diet did not show a significant difference from those fed an AsA-deficient diet (data not shown), mice fed a standard diet were used in the following experiments. Body weight and kidney weight did not differ significantly among these groups of mice at 9 weeks of age (Supplementary Figure 1 available online at http://informahealthcare.com/ doi/abs/10.3109/10715762.2014.915031). We then performed histological analyses of the kidneys and found them to be normal as judged by PAS and Elastica Masson staining (Figure 3). Plasma levels of BUN and creatinine were also within normal ranges (Supplementary Figure 2 available online at http://informahealthcare.com/ doi/abs/10.3109/10715762.2014.915031), indicating normal renal function in the KO mice despite low AsA levels. These data revealed that an Akr1a deficiency did not grossly affect renal structure and function under the breeding conditions up to 9 weeks of age.
1119
Histological examination of the kidneys after UUO Because AsA is a well-known cofactor for collagen synthesis via the donation of electrons to collagen prolyl hydroxylase [26], we examined the effects of an Akr1a deficiency on the pathogenesis of kidneys using a UUO model. Ureteral obstruction was achieved in the male mice at 9–10 weeks of age, because the kidneys of the male mice were more affected by UUO than those of the female mice. The young adult mice at this age did not show visible phenotypic damages at this point. Figure 4 shows the histological data of the mice kidneys on day 5 after the operation to induce UUO. A widening of the interstitial space and a dilatation of the tubules were noted in all areas. Also, fibrous material and inflammatory cell infiltration had increased in the interstitial space, particularly in the medulla, by comparison with untreated mice. However, there were no differences in the severity of these lesions among the WT, KO, and KO AsA groups of mice. Glomerular sclerosis was rarely observed in all three groups of the mice, and the differences were not significant. While a moderate infiltration of lymphocytes and neutrophils was observed in the medullae of the KO mice without AsA, only mild lymphocytic infiltration was seen in the WT and KO AsA mice. Because granulation was observed in the renal medullae of the UUO kidneys, we graded the granulation from 0 to 3 under Elastica Masson staining as follows: no (0), weak (1), moderate (2), and severe (3) granulation. The grade in the UUO kidneys of the KO mice tended to be higher than those in the WT and KO AsA mice, but again the differences
Figure 4. Histological examination of the kidneys of WT mice and KO mice with and without the administration of AsA after UUO. UUO operation was performed for WT, KO, and KO AsA mice. On day 5 after the operation to induce UUO, kidneys were dissected and the tissue sections were subjected to staining with PAS (upper panels) and Elastica Masson (lower panels). Typical data of the medulla and the cortex from several experiments are shown. Magnification: 200; Bars 100 μm.
1120 H. Nishida et al. (A)
WT UUO Day 5
KO − AsA
KO + AsA
AKR1A
Size (kDa) 35
AKR1B
35
TGF β
25
−
+
−
+
−
+
Collagen I 48
(B) 10 Collagen I (relative to WT sham)
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
αSMA
8 6 4
production of TGFβ and αSMA was observed in the UUO kidneys (Figure 5A), which was consistent with the proliferation of myofibroblasts in the damaged kidneys [27]. We also confirmed an increased production of collagen I in the UUO kidneys compared with the corresponding untreated kidneys. Unexpectedly, however, no significant differences were observed in the levels of collagen I among the WT, KO, and KO AsA groups of mice (Figures 5A and B) despite the established role of AsA in collagen formation. During fibrosis, proteins constituting a tissue matrix are produced within the ER of myofibroblasts, and are then secreted. Prolylhydroxylation with subsequent oxidative protein folding is essential for the secretion of collagen [26]. Therefore, we examined the levels of ER resident proteins PRDX4 and PDI, which are essential for oxidative protein folding in ER and for the presence of Bip, which is an ER chaperone. Immunoblot analyses showed that the expression of PRDX4 was highly induced in UUO kidneys in all experimental groups, while the increases in PDI and Bip were moderate (Figure 5C). Levels of 8-OHdG and antioxidative enzymes in the kidneys caused by UUO
2 0 UUO Day 5
−
+
−
+
−
KO − AsA
WT
+
KO + AsA
(C) UUO Day 5 PRDX4
WT −
KO − AsA +
−
+
KO + AsA −
+
Size (kDa) 25
PDI
Bip
75
Figure 5. Expression of marker proteins for fibrosis and ER-resident proteins in the kidneys of WT and KO mice. (A) The expressions of AKR1A, AKR1B, collagen I, and αSMA were detected by immunoblot analyses using corresponding antibodies. (B) Intensities of each band for collagen I were semi-quantified and values relative to those of a WT mouse without administration of AsA l were presented as the mean SEM for four mice. (C) Expressions of PRDX4, PDI, and Bip were detected by immunoblot analysis. Typical data from four experiments are shown.
were not significant (Supplementary Figure 3 available online at http://informahealthcare.com/doi/abs/10.3109/ 10715762.2014.915031). Increased expression of TGFβ, αSMA, and collagen I in the kidneys after UUO We then examined the expression of TGFβ, a principal factor for myofibroblast differentiation, αSMA, a protein marker for myofibroblast, and collagen I [27]. Induced
Because oxidative stress is likely to be an aggravating factor for UUO pathogenesis [7,9], we examined levels of an oxidative stress marker. Immunohistochemical analysis for 8-OHdG, which is a marker of oxidative damage in DNA [28], indicated weak positive staining in the nuclei of the control WT mice kidneys and a marked elevation by UUO (Figure 6). Nuclei in the renal tubules in both WT and KO were stained strongly positive in UUO conditions. When KO mice were administered AsA, the levels of the 8-OHdG decreased only slightly. We then examined the expression of major antioxidative enzymes SOD1, SOD2, GPX1, PRDX1, and catalase via immunoblot analyses (Figure 7). The GPX1 levels were markedly decreased by UUO while the levels of catalase and PRDX1 were only slightly decreased. The expression of SOD1, a cytosolic isoform, was also decreased by UUO, but the levels of SOD2, a mitochondriaspecific isoform, were more markedly decreased. Decrease in mitochondrial electron transport complexes in the kidneys after UUO NADPH oxidase from inflammatory cells appears to be a cause for ROS [7,9], but a potential target for ROS in the kidney cells is unclear. The decrease in SOD2 suggested the mitochondrial damage from UUO. In order to confirm the mitochondria as the likely target, we examined the protein levels of the major components of all five mitochondrial electron transport complexes (METC) and cyt c and found them to be markedly decreased on day 5 after the inducement of UUO (Figure 8A). We then tried to validate if mitochondrial damage was the deteriorating mechanism of UUO. We examined the time-dependent changes of mitochondrial proteins METC,
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Mitochondrial dysfunction in renal failure
1121
Figure 6. Immunohistological detection of 8-OHdG in the kidneys of WT and KO mice with and without the administration of AsA. Kidney sections of WT, KO, and KO AsA mice were reacted with anti-8-OHdG antibody. Typical data from several experiments are shown. Magnification: 400; Bars 50 μm.
cyt c, and SOD2 on 0, 1, 2, 3, 5, and 7 days after the initiation of UUO and compared them with those of fibrosis marker proteins collagen I and αSMA (Figure 8B). We found gradual decreases in all mitochondrial proteins from early time points after the initiation of UUO while the proteins related to fibrosis increased inversely. Thus, the mitochondrial damage preceded the development of fibrosis. WT − Catalase
GPX1
SOD2 SOD1
PRDX1
β-actin
+
KO − AsA
KO + AsA
−
−
+
+
Size (kDa) 50
20
20 15
20 48
Figure 7. Expression of antioxidative enzymes in the kidneys of WT and KO mice. Expressions of catalase, GPX1, SOD1, SOD2, PRDX1, and β-actin were detected by immunoblot analyses using corresponding antibodies. Typical data from four experiments are shown.
Discussion In the present study, we expected UUO to have an impact on the kidneys, but found no significant differences in the kidney damage among the WT, KO, and KO AsA mice groups (Figure 4 and Supplementary Figure 3 available online at http://informahealthcare.com/doi/abs/10.3109/ 10715762.2014.915031). Amazingly, UUO also caused no differences in collagen production among the WT, KO, and KO AsA groups (Figure 5), despite the established role of AsA in collagen synthesis [26]. Our observations are consistent with those of Parsons et al. [29] who reported normal collagen status in Gulo-deficient mice and suggested that electron donation from other sources might support collagen synthesis. Thus, other electron donors may compensate for the AsA deficiency in our UUO model, as well. Alternatively, Akr1a-deficient mice still produce about 10% AsA (Figure 2C) via an AKR1Bcatalyzed reaction [17], which might imply that such low levels of AsA are sufficient for collagen production in the kidneys of mice. Premature death and the rescue by AsA supplementation observed in Akr1a-deficient mice (Figure 1B) have also been reported in Gulo-deficient mice [29], indicating that the AsA production via AKR1B is not sufficient for the survival of Akr1a-deficient mice. Hence, it is likely that other electron donors support collagen formation in the kidney. During the process of fibrosis by UUO, ureteric epithelial cells and interstitial cells die constantly, and matrix proteins such as collagen are produced and fill the space for the dead cells [1,2]. Proapoptic genes are highly expressed in UUO kidneys, implying that cells are largely
1122 H. Nishida et al. (A)
WT
UUO Day 5 CV CIII CIV
–
KO –AsA +
–
+
KO + AsA –
+
Size (kDa) 48 35
CII 25 CI
17 11
cyt c
48
β-actin
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
PRDX1
20 UUO / WT
(B) UUO (days)
0
1
2
3
5
7
Size (kDa)
CV CIII CIV
48
CII
25
CI
cyt c SOD2 Collagen I αSMA
β-actin
PRDX1
35
17
11 25
63 48
48
20
Figure 8. Evaluation of mitochondrial damage in kidneys caused by UUO. (A) All mitochondrial electron transfer complexes and cyt c were detected by immunoblot analyses of proteins from the kidneys of WT and KO mice after UUO at day 5. (B) Timedependent changes of the METC, cyt c, SOD2, αSMA, and collagen I were examined by immunoblot analyses using corresponding antibodies. CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV; and, CV, complex V. Typical data from four experiments are shown.
lost due to apoptosis [30]. While TGFβ is a key promoting factor in fibrosis, myofibroblasts originate from various cell sources and characteristically express αSMA that produces matrix proteins of mainly collagen. Hence, they are directly involved in fibrosis [27]. We confirmed the induced expression of TGFβ and αSMA and the marked increase in collagen I in the kidneys, irrespective of the genotypes of the mice (Figure 5). Oxidative stress is a leading candidate for the cause of kidney cell death by UUO [6,7]. NADPH oxidase in the infiltrated inflammatory cells are regarded as a source of ROS in damaged kidneys [31]. We demonstrated an
elevated oxidative modification of DNA by detecting 8OH-dG (Figure 6), which is a hallmark of oxidative DNA damage [28]. In the present study, however, we found only moderate numbers of invaded inflammatory cells in the affected area of KO mice and rarely in WT and KO mice administered AsA. Thus, ROS derived from the inflammatory cells alone may not fully explain the constant cell death, but there could be another source for ROS. Mitochondrial electron transport systems are a wellknown source of ROS, and the mitochondrial dysfunction causes apoptotic cell death by releasing cyt c [32]. The results of the present study are the first to show how mitochondrial electron transport complexes were markedly decreased at an early period of renal damage by UUO (Figure 8). Improvement of the renal damage by suppressing mitochondria-derived ROS has been reported. For example, activation of Nrf-2 signaling by sulforaphane has preserved mitochondrial function and resulted in the suppression of UUO-induced oxidative stress and fibrosis [33]. A cell-permeable tetrapeptide that selectively targets the inner mitochondrial membrane suppresses oxidative stress and protects the kidneys from the damage elicited by UUO [34]. Thus, mitochondria appear to be a likely source for ROS because they consume most of the respired oxygen and generate ROS. We speculate that a defect in METC may occur primarily by UUO, which then triggers the production of ROS, which then impairs the antioxidative system, and consequently causes the death of interstitial cells and inflammatory reactions. Because we still do not know what directly causes the mitochondrial damage, this issue requires further examination. We also found decreases in the levels of antioxidative enzymes in the kidneys of WT and KO mice with and without AsA (Figure 7). Among the antioxidative enzymes, GPX1 exhibited the most marked decrease, which is consistent with the decreased GPX activity observed in kidneys with UUO [7]. GPX1 is a selenoprotein that contains a selenocysteine (SeCys) residue at the catalytic center. Nitric oxide that is induced by inflammation causes inactivation of GPX1 by oxidizing SeCys [35]. Hydrogen peroxide converts the SeCys to dehydroalanine and results in irreversible inactivation [36]. The conversion from SeCys to dehydroalanine in vivo is indeed demonstrated in the liver of SOD1deficient mice [37]. Although superoxide directly inactivates GPX1 [38], we have reported no changes in the levels of the GPX1 protein between SOD1-deficient and WT mice kidneys [39]. Thus, hydrogen peroxide, but not superoxide, appears to be involved in the pathogenesis of kidneys with UUO. We observed the elevated production of some proteins that reside in the ER lumen (Figure 5C). Accumulation of a large body of damaged proteins in the ER causes ER stress, which ultimately leads to cell death under extreme situations [40]. PRDX4 together with ERO1 appears to be required for collagen production because a deficiency decreases AsA and results in decreased collagen production [41]. However, it is unlikely that ER stress was the cause of renal cell death in the UUO model as follows.
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Mitochondrial dysfunction in renal failure
We observed that the expression of PRDX4, a protein thiol oxidase responsible for oxidative protein folding in the ER, was markedly induced in the kidneys by UUO, but that the levels of PDI and Bip, other hallmarks of ER stress, were increased only slightly (Figure 5C). Because PRDX4 also was secreted [42], the elevated levels of PRDX4 in the kidneys as a result of UUO may have reflected the presence of extracellular PRDX4. In fact, overexpressed PRDX4 ameliorates liver fibrosis by suppressing oxidative stress [43]. If ER stress were the underlying mechanism for the renal damage, collagen production would conversely be decreased due to the dysfunction of the ER. Thus, the elevation in PRDX4 together with other ER resident proteins appears to have been an adaptive response of myofibroblasts to activate ER function for collagen secretion. In conclusion, it was unexpected that no significant difference was observed in collagen production as a result of UUO, irrespective of the amounts of AsA in the mice, which implies either a low demand for AsA or the presence of another electron donor for collagen production in the kidneys. A marked induction of PRDX4 was observed, which would imply an activated ER function in myofibroblasts for the secretion of a large body of collagen. Mitochondrial damage, which occurred at the initial stage of kidney dysfunction, appeared to be a cause of ROS elevation and a further triggering of cell death and fibrosis.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
References [1] Klahr S, Morrissey J. Obstructive nephropathy and renal fibrosis. Am J Physiol Renal Physiol 2002;283:F861–F875. [2] Bascands JL, Schanstra JP. Obstructive nephropathy: insights from genetically engineered animals. Kidney Int 2005;68: 925–937. [3] Schreiner GF, Harris KP, Purkerson ML, Klahr S. Immunological aspects of acute ureteral obstruction: immune cell infiltrate in the kidney. Kidney Int 1988;34:487–493. [4] Cachat F, Lange-Sperandio B, Chang AY, Kiley SC, Thornhill BA, Forbes MS, Chevalier RL. Ureteral obstruction in neonatal mice elicits segment-specific tubular cell responses leading to nephron loss. Kidney Int 2003;63:564–575. [5] Rouschop KM, Claessen N, Pals ST, Weening JJ, Florquin S. CD44 disruption prevents degeneration of the capillary network in obstructive nephropathy via reduction of TGFbeta1-induced apoptosis. J Am Soc Nephrol 2006;17:746–753. [6] Kawada N, Moriyama T, Ando A, Fukunaga M, Miyata T, Kurokawa K, et al. Increased oxidative stress in mouse kidneys with unilateral ureteral obstruction. Kidney Int 1999; 56:1004–1013. [7] Kinter M, Wolstenholme JT, Thornhill BA, Newton EA, McCormick ML, Chevalier RL. Unilateral ureteral obstruction impairs renal antioxidant enzyme activation during sodium depletion. Kidney Int 1999;55:1327–1334.
1123
[8] Sunami R, Sugiyama H, Wang DH, Kobayashi M, Maeshima Y, Yamasaki Y, et al. Acatalasemia sensitizes renal tubular epithelial cells to apoptosis and exacerbates renal fibrosis after unilateral ureteral obstruction. Am J Physiol Renal Physiol 2004;286:F1030–F1038. [9] Dendooven A, Ishola DA Jr, Nguyen TQ, Van der Giezen DM, Kok RJ, Goldschmeding R, Joles JA. Oxidative stress in obstructive nephropathy. Int J Exp Pathol 2011;92:202–210. [10] Fujii J, Ito JI, Zhang X, Kurahashi T. Unveiling the roles of the glutathione redox system in vivo by analyzing genetically modified mice. J Clin Biochem Nutr 2011;49:70–78. [11] Du J, Cullen JJ, Buettner GR. Ascorbic acid: chemistry, biology and the treatment of cancer. Biochim Biophys Acta 2012;1826:443–457. [12] Linster CL, Van Schaftingen E. Vitamin C. Biosynthesis, recycling and degradation in mammals. FEBS J 2007;274: 1–22. [13] Mandl J, Szarka A, Bánhegyi G. Vitamin C: update on physiology and pharmacology. Br J Pharmacol 2009;157: 1097–1110. [14] Deicher R, Hörl WH. Vitamin C in chronic kidney disease and hemodialysis patients. Kidney Blood Press Res 2003; 26:100–106. [15] Coombes JS, Fassett RG. Antioxidant therapy in hemodialysis patients: a systematic review. Kidney Int 2012;81:233–246. [16] Sadat U, Usman A, Gillard JH, Boyle JR. Does ascorbic acid protect against contrast-induced acute kidney injury in patients undergoing coronary angiography: a systematic review with meta-analysis of randomized controlled trials. J Am Coll Cardiol 2013;62;2167–2175. [17] Gabbay KH, Bohren KM, Morello R, Bertin T, Liu J, Vogel P. Ascorbate synthesis pathway: dual role of ascorbate in bone homeostasis. J Biol Chem 2010;285:19510–19520. [18] Takahashi M, Miyata S, Fujii J, Inai Y, Ueyama S, Araki M, et al. In vivo role of aldehyde reductase. Bichim Biophys Acta 2012;1820:1787–1796. [19] Ito J, Otsuki N, Zhang X, Konno K, Kurahashi T, Takahashi M, et al. Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice. Life Sci 2014;95:1–8. [20] Takahashi M, Fujii J, Miyoshi E, Hoshi A, Taniguchi N. Elevation of aldose reductase gene expression in rat primary hepatoma and hepatoma cell lines: implication in detoxification of cytotoxic aldehydes. Int J Cancer 1995;62:749–754. [21] Matsuoka Y, Yamato M, Yamasaki T, Mito F, Yamada K. Rapid and convenient detection of ascorbic acid using a fluorescent nitroxide switch. Free Radic Biol Med 2012; 53:2112–2118. [22] Takahashi M, Fujii J, Teshima T, Suzuki K, Shiba T, Taniguchi N. Identity of a major 3-deoxyglucosone-reducing enzyme with aldehyde reductase in rat liver established by amino acid sequencing and cDNA expression. Gene 1993; 127:249–253. [23] Iuchi Y, Okada F, Onuma K, Onoda T, Asao H, Kobayashi M, Fujii J. Elevated oxidative stress in erythrocytes due to an SOD1 deficiency causes anemia and triggers autoantibody production. Biochem J 2007;402:219–227. [24] Ito R, Takahashi M, Ihara H, Tsukamoto H, Fujii J, Ikeda Y. Measurement of peroxiredoxin-4 serum levels in rat tissue and its use as a potential marker for hepatic disease. Mol Med Rep 2012;6:379–384. [25] Fujii T, Endo T, Fujii J, Taniguchi N. Differential expression of glutathione reductase and cytosolic glutathione peroxidase, GPX1, in developing rat lungs and kidneys. Free Radic Res 2002;36:1041–1049. [26] Ishikawa Y, Bächinger HP. A molecular ensemble in the rER for procollagen maturation. Biochim Biophys Acta 2013; 1833:2479–2491. [27] Hinz B, Phan SH, Thannickal VJ, Prunotto M, Desmoulière A, Varga J, et al. Recent developments in myofibroblast biology:
1124 H. Nishida et al.
[28] [29] [30]
[31]
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
[32] [33]
[34]
[35]
paradigms for connective tissue remodeling. Am J Pathol 2012;180:1340–1355. Toyokuni S, Mori T, Dizdaroglu M. DNA base modifications in renal chromatin of Wistar rats treated with a renal carcinogen, ferric nitrilotriacetate. Int J Cancer 1994;57:123–128. Parsons KK, Maeda N, Yamauchi M, Banes AJ, Koller BH. Ascorbic acid-independent synthesis of collagen in mice. Am J Physiol Endocrinol Metab 2006;290:E1131–E1139. Forbes MS, Thornhill BA, Chevalier RL. Proximal tubular injury and rapid formation of atubular glomeruli in mice with unilateral ureteral obstruction: a new look at an old model. Am J Physiol Renal Physiol 2011;301:F110–F117. Barnes JL, Gorin Y. Myofibroblast differentiation during fibrosis: role of NAD(P)H oxidases. Kidney Int 2011;79: 944–956. Mailloux RJ, McBride SL, Harper ME. Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics. Trends Biochem Sci 2013;38:592–602. Chung SD, Lai TY, Chien CT, Yu HJ. Activating Nrf-2 signaling depresses unilateral ureteral obstruction-evoked mitochondrial stress-related autophagy, apoptosis and pyroptosis in kidney. PLoS One 2012;7:e47299. Mizuguchi Y, Chen J, Seshan SV, Poppas DP, Szeto HH, Felsen D. A novel cell-permeable antioxidant peptide decreases renal tubular apoptosis and damage in unilateral ureteral obstruction. Am J Physiol Renal Physiol 2008;295:F1545–1553. Asahi M, Fujii J, Takao T, Kuzuya T, Hori M, Shimonishi Y, Taniguchi N. The oxidation of selenocysteine is involved in the inactivation of glutathione peroxidase by nitric oxide donor. J Biol Chem 1997;272:19152–19157.
Supplementary material available online Supplementary Figures 1–3.
[36] Cho CS, Lee S, Lee GT, Woo HA, Choi EJ, Rhee SG. Irreversible inactivation of glutathione peroxidase 1 and reversible inactivation of peroxiredoxin II by H2O2 in red blood cells. Antioxid Redox Signal 2010;12:1235–1246. [37] Wang SK, Weaver JD, Zhang S, Lei XG. Knockout of SOD1 promotes conversion of selenocysteine to dehydroalanine in murine hepatic GPX1 protein. Free Radic Biol Med 2011;51:197–204. [38] Blum J, Fridovich I. Inactivation of glutathione peroxidase by superoxide radical. Arch Biochem Biophys 1985;240: 500–508. [39] Yamanobe T, Okada F, Iuchi Y, Onuma K, Tomita Y, Fujii J. Deterioration of ischemia/reperfusion-induced acute renal failure in SOD1-deficient mice. Free Radic Res 2007;41: 200–207. [40] Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science 2011;334: 1081–1086. [41] Zito E, Hansen HG, Yeo GS, Fujii J, Ron D. Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice. Mol Cell 2012; 48:39–51. [42] Okado-Matsumoto A, Matsumoto A, Fujii J, Taniguchi N. Peroxiredoxin IV is a secretable protein with heparin-binding properties under reduced conditions. J Biochem 2000;127: 493–501. [43] Nabeshima A, Yamada S, Guo X, Tanimoto A, Wang KY, Shimajiri S, et al. Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model. Antioxid Redox Signal 2013;19:1983–1998.
ISSN: 1071-5762 (Print) 1029-2470 (Online) Journal homepage: http://www.tandfonline.com/loi/ifra20
Kidney fibrosis is independent of the amount of ascorbic acid in mice with unilateral ureteral obstruction H. Nishida, T. Kurahashi, Y. Saito, N. Otsuki, M. Kwon, H. Ohtake, M. Yamakawa, K.-I. Yamada, S. Miyata, Y. Tomita & J. Fujii To cite this article: H. Nishida, T. Kurahashi, Y. Saito, N. Otsuki, M. Kwon, H. Ohtake, M. Yamakawa, K.-I. Yamada, S. Miyata, Y. Tomita & J. Fujii (2014) Kidney fibrosis is independent of the amount of ascorbic acid in mice with unilateral ureteral obstruction, Free Radical Research, 48:9, 1115-1124 To link to this article: http://dx.doi.org/10.3109/10715762.2014.915031
View supplementary material
Accepted online: 15 Apr 2014.Published online: 21 Jul 2014.
Submit your article to this journal
Article views: 80
View related articles
View Crossmark data
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ifra20 Download by: [Karolinska Institutet, University Library]
Date: 12 September 2015, At: 16:30
Free Radical Research, September 2014; 48(9): 1115–1124 © 2014 Informa UK, Ltd. ISSN 1071-5762 print/ISSN 1029-2470 online DOI: 10.3109/10715762.2014.915031
ORIGINAL ARTICLE
Kidney fibrosis is independent of the amount of ascorbic acid in mice with unilateral ureteral obstruction H. Nishida1,2, T. Kurahashi1, Y. Saito1, N. Otsuki1, M. Kwon1, H. Ohtake3, M. Yamakawa3, K.-I. Yamada4, S. Miyata5, Y. Tomita2 & J. Fujii1 1Department
of Biochemistry and Molecular Biology, Yamagata University School of Medicine, Yamagata, Japan, of Urology, Yamagata University School of Medicine, Yamagata, Japan, 3Department of Diagnostic Pathology, Faculty of Medicine, Yamagata University, Yamagata, Japan, 4Department of Bio-functional Science, Faculty of Pharmacological Science, Kyushu University, Fukuoka, Japan, and 5Department of Internal Medicine, Osaka Koseinenkin Hospital, Osaka, Japan
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
2Department
Abstract In response to sustained damage to a kidney, fibrosis that can be characterized as the deposition of a collagenous matrix occurs and consequently causes chronic kidney failure. Because most animals used in experiments synthesize ascorbic acid (AsA) from glucose, the roles of AsA in fibrotic kidney diseases are largely unknown. Unilateral ureteric obstruction (UUO) mimics the complex pathophysiology of chronic obstructive nephropathy and is an ideal model for the investigation of the roles of AsA in kidney failure. We examined the impact of a deficiency of Akr1a, a gene that encodes aldehyde reductase and is responsible for the production of AsA, on fibrotic damage caused by UUO in mice. Oxidatively modified DNA was elevated in wild-type and Akr1a-deficient kidneys as a result of UUO to a similar extent, and was only slightly suppressed by the administration of AsA. Even though Akrla-deficient mice could produce only about 10% of the AsA produced by wild-type mice, no difference was observed in collagen I synthesis under pathological conditions. The data implied either a low demand for AsA or the presence of another electron donor for collagen I production in the mouse kidney. Next, we attempted to elucidate the potential causes for oxidative damage in kidney cells during the fibrotic change. We found decreases in mitochondrial proteins, particularly in electron transport complexes, at the initial stage of the kidney fibrosis. The data imply that a dysfunction of the mitochondria leads to an elevation of ROS, which results in kidney fibrosis by stimulating cellular transformation to myofibroblasts. Keywords: mitochondria, kidney, oxidative stress
Introduction In response to sustained damage to a kidney, fibrosis characterized as the deposition of a collagenous matrix occurs and consequently causes chronic kidney failure. Unilateral ureteric obstruction (UUO) is an experimental model of renal injury that mimics the complex pathophysiology of chronic obstructive nephropathy [1,2]. The hydrostatic pressure induced by UUO leads to tubular cell death, interstitial inflammatory infiltration, capillary rarefaction, loss of renal parenchyma, and progressive fibrosis by activating myofibroblasts [3–5]. In this condition, oxidative stress markers such as malondialdehyde, 8-hydroxyguanine (8-OHdG), and 8-isoprostaglandin F2αl also are elevated [6,7], and mice that lack catalase are more susceptible to UUO-induced renal injury compared with wild-type mice [8]. Thus, oxidative stress is involved in the pathogenesis of UUO, and NADPH oxidase from inflammatory cells has been implicated as the potential source for reactive oxygen species (ROS) [9]. Other than catalase, antioxidative enzymes that include superoxide dismutase (SOD), glutathione peroxidase (GPX), and peroxiredoxin play roles in suppressing oxidative injury during the inflammatory process and in other
diseases. In addition to the enzymes, low molecular weight compounds such as glutathione and vitamin C (ascorbic acid; AsA) also function to maintain the redox balance of the cells by eliminating ROS [10,11]. On the other hand, AsA is an important cofactor for some oxygenases and prolyl hydroxylase and participates in a variety of physiological reactions such as collagen synthesis, adrenaline production, and steroidogenesis [12,13]. The nephroprotective effects of AsA have been documented under some disease conditions [14–16], although ambiguity remains from a clinical point of view. While most vertebrates including rodents can naturally synthesize AsA, humans cannot due to a loss of function of L-gulono-γ-lactone oxidase (Gulo) that localizes in the endoplasmic reticulum (ER) membrane and catalyzes the last step of AsA synthesis [13]. Recently our group and others have found that aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, is the enzyme responsible for the production of AsA by reducing D-glucuronic acid in mice [17,18]. Aldose reductase (AKR1B), a closely related family member, also catalyzes the same reaction although its contribution to AsA synthesis is ∼15% in the mouse body [17].
Correspondence: Junichi Fujii, Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Yamagata University, 2-2-2 Iidanishi, Yamagata 990-9585, Japan. Tel: 81-23-628-5227. Fax: 81-23-628-5230. E-mail: [email protected] (Received date: 27 January 2014; Accepted date: 10 April 2014; Published online: 12 May 2014)
1116 H. Nishida et al. Here, we examined whether Akr1a deficiency affects collagen synthesis under the pathological conditions elicited by UUO. We also found mitochondrial dysfunction to be a likely cause for ROS production.
Experimental
Akr1a knockout (KO) mice were generated using the gene-targeting technique and were genotyped by PCR, as described in the literature [18]. After backcrosses to C57BL/6 N mice for more than 8 times, Akr1a/ (WT) and Akr1a/ (KO) littermates were obtained [19]. The WT and KO mice were weaned at 30 days of age and fed either an AsA-deficient diet (CL-2; CLEA Japan, Tokyo) or a standard diet (Picolab 5053, LabDiet, St Louis, MO) ad libitum with free access to either water or water containing 1.5 mg/ml AsA until use. All animal experiments were performed in accordance with the Declaration of Helsinki under the protocol approved by the Animal Research Committee of this institution.
Blood test Blood was collected in the presence of excess ethylenediaminetetraacetic acid (EDTA) from the tail vein at the time of ureter ligation and from the inferior vena cava at the time of kidney harvest. After centrifugation, the plasma concentrations of blood urea nitrogen (BUN) and creatinine were determined using Fuji Drychem 3500 V and Fuji Dry-chem slides (Fuji film), respectively.
(A)
50
Surgical operation and experimental design
Histological and immunohistochemical analyses Tissue sections 2 to 3 μm in thickness were rehydrated and subjected to periodic acid-Schiff (PAS) and Elastica Masson staining for the evaluation of kidney injury. Rehydrated tissue sections were also subjected to immunohistochemical analysis. Briefly, we incubated the tissue sections with 3% hydrogen peroxide solution to eliminate intrinsic peroxidase activity. The target retrieval procedure involved the immersion of tissue sections in a citrate-based buffer solution followed by heating in an autoclave. After rinsing, the sections were incubated for 10 min with 2% BSA/TBS at room temperature, followed by incubation with a rabbit anti-rat AKR1A polyclonal antibody [20] and a mouse anti-8-hydroxydeoxyguanine
WT KO KO+ASA
40 Body weight (g)
Male mice were anesthetized via the inhalation of isoflurane [19]. Ureteral obstruction was achieved on male mice at 9–10 weeks of age by ligating the left proximal ureter with 4-0 silk through a left lateral incision. The mice were euthanized on either 5 days in most experiments or 0, 1, 2, 3, 5, and 7 days in a time course experiment after surgical operation under ether anesthesia, and the kidneys were harvested for evaluation. The right-side kidneys of the mice not subjected to the operation were also harvested for control samples. The harvested kidneys were bisected just after excision. One-half of the kidneys was frozen in liquid nitrogen immediately, and then it was stored at –80°C for determination of proteins and lipid peroxidation. The other half was fixed in 10% buffered formalin followed by embedding in paraffin.
30
20
10 8
(B)
16
24
32 40 Age (weeks)
48
56
100
80 Survival rate (%)
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Animals
(8-OHdG) monoclonal antibody (MOG-100 at a concentration of 5 μg/ml; Nikken Seil, Shizuoka) overnight in a humidified chamber at 4°C. After rinsing, the sections were sequentially reacted with peroxidase-labeled goat anti-rabbit and mouse IgG polymer for 30 min. After a final rinse, specific immunolabeling was visualized by placing a chromogen, 3,3′-diaminobenzine (K1391, Dako, USA) on the tissue sections for a few minutes. The sections were then dehydrated, mounted, and examined via light microscopy.
60
40 WT
20
KO KO+AsA
0 0
10
20
30
40
50
Age (weeks)
Figure 1. Changes in the body weight and survival rates of KO mice with and without the administration of AsA. (A) Age-dependent changes in the body weight of the WT, KO, and KO AsA mice. (B) Kaplan–Meier analysis was performed for each group. Mean SEM for 10 female mice.
Mitochondrial dysfunction in renal failure
Measurement of plasma ascorbic acid
curve constructed using 0, 10, 25, 50, 75, 100 μM of AsA. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis Kidneys were homogenized in RIPA Buffer and centrifuged at 17 400 g in a microcentrifuge. The supernatant was used for protein assay via a BCA protein assay reagent (Thermo Fisher Scientific, Yokohama, Japan). The protein samples (30 or 40 μg) were subjected to 7.5–15% sodium dodecylsulfate-polyacrylamide gel electrophoresis
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
To detect the AsA in plasma, 5 μl of plasma was added to an assay solution containing 85 μl of distilled water and 10 μl of 500 μM 15-(naphthalen-1-ylamino)-7aza-3,11-dioxadispiro[5.1.5 8 .3 6 ]hexadecan-7 - oxyl (Naph-DiPy) fluorescent indicator [21]. After a 30-min reaction at room temperature, the fluorescence was measured at an excitation wavelength of 310 nm and an emission wavelength of 430 nm using a Valioskan Flash microplate reader (Thermo Fisher Scientific). The concentrations were calculated from a standard
1117
Figure 2. Expression of AKR1A in kidneys and plasma levels of AsA in WT and KO mice. (A) Expression of AKR1A and AKR1B was detected by immunoblot analysis using anti-AKR1A antibody. (B) Immunohistochemical detection of AKR1A in the kidneys of WT and KO mice. Typical data from several experiments were shown. 400. Bars 50 μm. (C) Plasma levels of AsA were measured using a fluorescent indicator. Means SD are shown.
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
1118 H. Nishida et al. (SDS-PAGE) and electroblotted onto PVDF membranes (GE-Health care). The membranes were then blocked by incubation with 3% skimmed milk in TBST (150 mM NaCl and 20 mM Tris/HCl, pH7.6, 0.1% Tween-20) for 1 h at room temperature. The membranes were then incubated with antibodies to AKR1A [22], aldose reductase (AKR1B) [20], SOD1 [23], SOD2 [23], PRDX1 [23], PRDX4 [24], glutathione peroxidase 1 (GPX1) [25], catalase (219010, Carbiochem-Merck Bioscience, Billerica, MA, USA), cytochrome c (cyt c; Pharmingen), the mouse monoclonal antibody against β-actin (sc-69879, Santa Cruz Biotechnology), or a total Oxphos rodent antibody cocktail that included five antibodies against mitochondrial electron transfer complexes (METC) (ab11013, Abcam). After washing with TBST, the membrane was incubated with a 1:20 000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz Biotechnology) for 1 h at room temperature. After washing with TBST, the immunoreactive bands were detected by measuring chemiluminescence using Immobilon Western (Millipore) on an image analyzer (ImageQuant LAS500, GE Healthcare). Each protein amount was semi-quantified using NIH image software, and the relative values are shown.
test followed by a Tukey–Kramer multiple comparison test. A p-value of less than 0.05 was considered significant.
Statistical analysis
AKR1A expression and localization in the mouse kidney
The results are expressed as the mean SD. Comparison between groups was performed using a one-way ANOVA
We examined the expression and localization of AKR1A in the kidneys of WT and KO mice. While the kidneys of
Results AsA deficiency causes weight loss and premature death in aged AKR1A-KO mice We first investigated the body weight and the lifespan of KO mice and compared them with those of WT mice and KO mice supplemented with AsA in drinking water (KO AsA). The body weight of the KO mice was the same as the mice in the other group up to ∼20 weeks, at which time it decreased (Figure 1A). The KO mice started dying around 10 weeks of age and all had died within 1 year under the AsA-deficient diet (Figure 1B). However, supplementation of AsA (1.5 mg/ml in drinking water) contributed to maintaining body weight and extended the life-span of the KO mice. This suggests that the deaths were caused mainly by AsA deficiency due to the lack of AKR1A and that the lives of these mice could not have been supported by the AsA that was produced via AKR1B function alone.
Figure 3. Histological appearance of the kidneys of WT and KO mice with and without the administration of AsA. Kidney sections of WT, KO, and KO AsA mice were stained with PAS (upper panels) or Elastica Masson (lower panels). Magnification: 100; Bars 200 μm.
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Mitochondrial dysfunction in renal failure
the WT mice showed bands at corresponding positions of AKR1A and AKR1B, no detectable AKR1A proteins were observed in the KO kidneys (Figure 2A). Immunohistochemical staining using the anti-AKR1A antibody revealed that AKR1A was highly expressed in the epithelial cells lining the Bowman’s capsules and proximal tubules, particularly on the brush border (Figure 2B). Because AKR1A is involved in AsA synthesis [17,18], we measured the plasma levels of AsA in WT mice, KO mice, and KO mice supplemented with AsA in drinking water (KO AsA) and found these levels initially to be very low in the KO mice compared with the WT mice, but these levels recovered to wild-type levels after AsA supplementation (Figure 2C). Because AsA levels in the KO mice fed a standard diet did not show a significant difference from those fed an AsA-deficient diet (data not shown), mice fed a standard diet were used in the following experiments. Body weight and kidney weight did not differ significantly among these groups of mice at 9 weeks of age (Supplementary Figure 1 available online at http://informahealthcare.com/ doi/abs/10.3109/10715762.2014.915031). We then performed histological analyses of the kidneys and found them to be normal as judged by PAS and Elastica Masson staining (Figure 3). Plasma levels of BUN and creatinine were also within normal ranges (Supplementary Figure 2 available online at http://informahealthcare.com/ doi/abs/10.3109/10715762.2014.915031), indicating normal renal function in the KO mice despite low AsA levels. These data revealed that an Akr1a deficiency did not grossly affect renal structure and function under the breeding conditions up to 9 weeks of age.
1119
Histological examination of the kidneys after UUO Because AsA is a well-known cofactor for collagen synthesis via the donation of electrons to collagen prolyl hydroxylase [26], we examined the effects of an Akr1a deficiency on the pathogenesis of kidneys using a UUO model. Ureteral obstruction was achieved in the male mice at 9–10 weeks of age, because the kidneys of the male mice were more affected by UUO than those of the female mice. The young adult mice at this age did not show visible phenotypic damages at this point. Figure 4 shows the histological data of the mice kidneys on day 5 after the operation to induce UUO. A widening of the interstitial space and a dilatation of the tubules were noted in all areas. Also, fibrous material and inflammatory cell infiltration had increased in the interstitial space, particularly in the medulla, by comparison with untreated mice. However, there were no differences in the severity of these lesions among the WT, KO, and KO AsA groups of mice. Glomerular sclerosis was rarely observed in all three groups of the mice, and the differences were not significant. While a moderate infiltration of lymphocytes and neutrophils was observed in the medullae of the KO mice without AsA, only mild lymphocytic infiltration was seen in the WT and KO AsA mice. Because granulation was observed in the renal medullae of the UUO kidneys, we graded the granulation from 0 to 3 under Elastica Masson staining as follows: no (0), weak (1), moderate (2), and severe (3) granulation. The grade in the UUO kidneys of the KO mice tended to be higher than those in the WT and KO AsA mice, but again the differences
Figure 4. Histological examination of the kidneys of WT mice and KO mice with and without the administration of AsA after UUO. UUO operation was performed for WT, KO, and KO AsA mice. On day 5 after the operation to induce UUO, kidneys were dissected and the tissue sections were subjected to staining with PAS (upper panels) and Elastica Masson (lower panels). Typical data of the medulla and the cortex from several experiments are shown. Magnification: 200; Bars 100 μm.
1120 H. Nishida et al. (A)
WT UUO Day 5
KO − AsA
KO + AsA
AKR1A
Size (kDa) 35
AKR1B
35
TGF β
25
−
+
−
+
−
+
Collagen I 48
(B) 10 Collagen I (relative to WT sham)
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
αSMA
8 6 4
production of TGFβ and αSMA was observed in the UUO kidneys (Figure 5A), which was consistent with the proliferation of myofibroblasts in the damaged kidneys [27]. We also confirmed an increased production of collagen I in the UUO kidneys compared with the corresponding untreated kidneys. Unexpectedly, however, no significant differences were observed in the levels of collagen I among the WT, KO, and KO AsA groups of mice (Figures 5A and B) despite the established role of AsA in collagen formation. During fibrosis, proteins constituting a tissue matrix are produced within the ER of myofibroblasts, and are then secreted. Prolylhydroxylation with subsequent oxidative protein folding is essential for the secretion of collagen [26]. Therefore, we examined the levels of ER resident proteins PRDX4 and PDI, which are essential for oxidative protein folding in ER and for the presence of Bip, which is an ER chaperone. Immunoblot analyses showed that the expression of PRDX4 was highly induced in UUO kidneys in all experimental groups, while the increases in PDI and Bip were moderate (Figure 5C). Levels of 8-OHdG and antioxidative enzymes in the kidneys caused by UUO
2 0 UUO Day 5
−
+
−
+
−
KO − AsA
WT
+
KO + AsA
(C) UUO Day 5 PRDX4
WT −
KO − AsA +
−
+
KO + AsA −
+
Size (kDa) 25
PDI
Bip
75
Figure 5. Expression of marker proteins for fibrosis and ER-resident proteins in the kidneys of WT and KO mice. (A) The expressions of AKR1A, AKR1B, collagen I, and αSMA were detected by immunoblot analyses using corresponding antibodies. (B) Intensities of each band for collagen I were semi-quantified and values relative to those of a WT mouse without administration of AsA l were presented as the mean SEM for four mice. (C) Expressions of PRDX4, PDI, and Bip were detected by immunoblot analysis. Typical data from four experiments are shown.
were not significant (Supplementary Figure 3 available online at http://informahealthcare.com/doi/abs/10.3109/ 10715762.2014.915031). Increased expression of TGFβ, αSMA, and collagen I in the kidneys after UUO We then examined the expression of TGFβ, a principal factor for myofibroblast differentiation, αSMA, a protein marker for myofibroblast, and collagen I [27]. Induced
Because oxidative stress is likely to be an aggravating factor for UUO pathogenesis [7,9], we examined levels of an oxidative stress marker. Immunohistochemical analysis for 8-OHdG, which is a marker of oxidative damage in DNA [28], indicated weak positive staining in the nuclei of the control WT mice kidneys and a marked elevation by UUO (Figure 6). Nuclei in the renal tubules in both WT and KO were stained strongly positive in UUO conditions. When KO mice were administered AsA, the levels of the 8-OHdG decreased only slightly. We then examined the expression of major antioxidative enzymes SOD1, SOD2, GPX1, PRDX1, and catalase via immunoblot analyses (Figure 7). The GPX1 levels were markedly decreased by UUO while the levels of catalase and PRDX1 were only slightly decreased. The expression of SOD1, a cytosolic isoform, was also decreased by UUO, but the levels of SOD2, a mitochondriaspecific isoform, were more markedly decreased. Decrease in mitochondrial electron transport complexes in the kidneys after UUO NADPH oxidase from inflammatory cells appears to be a cause for ROS [7,9], but a potential target for ROS in the kidney cells is unclear. The decrease in SOD2 suggested the mitochondrial damage from UUO. In order to confirm the mitochondria as the likely target, we examined the protein levels of the major components of all five mitochondrial electron transport complexes (METC) and cyt c and found them to be markedly decreased on day 5 after the inducement of UUO (Figure 8A). We then tried to validate if mitochondrial damage was the deteriorating mechanism of UUO. We examined the time-dependent changes of mitochondrial proteins METC,
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Mitochondrial dysfunction in renal failure
1121
Figure 6. Immunohistological detection of 8-OHdG in the kidneys of WT and KO mice with and without the administration of AsA. Kidney sections of WT, KO, and KO AsA mice were reacted with anti-8-OHdG antibody. Typical data from several experiments are shown. Magnification: 400; Bars 50 μm.
cyt c, and SOD2 on 0, 1, 2, 3, 5, and 7 days after the initiation of UUO and compared them with those of fibrosis marker proteins collagen I and αSMA (Figure 8B). We found gradual decreases in all mitochondrial proteins from early time points after the initiation of UUO while the proteins related to fibrosis increased inversely. Thus, the mitochondrial damage preceded the development of fibrosis. WT − Catalase
GPX1
SOD2 SOD1
PRDX1
β-actin
+
KO − AsA
KO + AsA
−
−
+
+
Size (kDa) 50
20
20 15
20 48
Figure 7. Expression of antioxidative enzymes in the kidneys of WT and KO mice. Expressions of catalase, GPX1, SOD1, SOD2, PRDX1, and β-actin were detected by immunoblot analyses using corresponding antibodies. Typical data from four experiments are shown.
Discussion In the present study, we expected UUO to have an impact on the kidneys, but found no significant differences in the kidney damage among the WT, KO, and KO AsA mice groups (Figure 4 and Supplementary Figure 3 available online at http://informahealthcare.com/doi/abs/10.3109/ 10715762.2014.915031). Amazingly, UUO also caused no differences in collagen production among the WT, KO, and KO AsA groups (Figure 5), despite the established role of AsA in collagen synthesis [26]. Our observations are consistent with those of Parsons et al. [29] who reported normal collagen status in Gulo-deficient mice and suggested that electron donation from other sources might support collagen synthesis. Thus, other electron donors may compensate for the AsA deficiency in our UUO model, as well. Alternatively, Akr1a-deficient mice still produce about 10% AsA (Figure 2C) via an AKR1Bcatalyzed reaction [17], which might imply that such low levels of AsA are sufficient for collagen production in the kidneys of mice. Premature death and the rescue by AsA supplementation observed in Akr1a-deficient mice (Figure 1B) have also been reported in Gulo-deficient mice [29], indicating that the AsA production via AKR1B is not sufficient for the survival of Akr1a-deficient mice. Hence, it is likely that other electron donors support collagen formation in the kidney. During the process of fibrosis by UUO, ureteric epithelial cells and interstitial cells die constantly, and matrix proteins such as collagen are produced and fill the space for the dead cells [1,2]. Proapoptic genes are highly expressed in UUO kidneys, implying that cells are largely
1122 H. Nishida et al. (A)
WT
UUO Day 5 CV CIII CIV
–
KO –AsA +
–
+
KO + AsA –
+
Size (kDa) 48 35
CII 25 CI
17 11
cyt c
48
β-actin
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
PRDX1
20 UUO / WT
(B) UUO (days)
0
1
2
3
5
7
Size (kDa)
CV CIII CIV
48
CII
25
CI
cyt c SOD2 Collagen I αSMA
β-actin
PRDX1
35
17
11 25
63 48
48
20
Figure 8. Evaluation of mitochondrial damage in kidneys caused by UUO. (A) All mitochondrial electron transfer complexes and cyt c were detected by immunoblot analyses of proteins from the kidneys of WT and KO mice after UUO at day 5. (B) Timedependent changes of the METC, cyt c, SOD2, αSMA, and collagen I were examined by immunoblot analyses using corresponding antibodies. CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV; and, CV, complex V. Typical data from four experiments are shown.
lost due to apoptosis [30]. While TGFβ is a key promoting factor in fibrosis, myofibroblasts originate from various cell sources and characteristically express αSMA that produces matrix proteins of mainly collagen. Hence, they are directly involved in fibrosis [27]. We confirmed the induced expression of TGFβ and αSMA and the marked increase in collagen I in the kidneys, irrespective of the genotypes of the mice (Figure 5). Oxidative stress is a leading candidate for the cause of kidney cell death by UUO [6,7]. NADPH oxidase in the infiltrated inflammatory cells are regarded as a source of ROS in damaged kidneys [31]. We demonstrated an
elevated oxidative modification of DNA by detecting 8OH-dG (Figure 6), which is a hallmark of oxidative DNA damage [28]. In the present study, however, we found only moderate numbers of invaded inflammatory cells in the affected area of KO mice and rarely in WT and KO mice administered AsA. Thus, ROS derived from the inflammatory cells alone may not fully explain the constant cell death, but there could be another source for ROS. Mitochondrial electron transport systems are a wellknown source of ROS, and the mitochondrial dysfunction causes apoptotic cell death by releasing cyt c [32]. The results of the present study are the first to show how mitochondrial electron transport complexes were markedly decreased at an early period of renal damage by UUO (Figure 8). Improvement of the renal damage by suppressing mitochondria-derived ROS has been reported. For example, activation of Nrf-2 signaling by sulforaphane has preserved mitochondrial function and resulted in the suppression of UUO-induced oxidative stress and fibrosis [33]. A cell-permeable tetrapeptide that selectively targets the inner mitochondrial membrane suppresses oxidative stress and protects the kidneys from the damage elicited by UUO [34]. Thus, mitochondria appear to be a likely source for ROS because they consume most of the respired oxygen and generate ROS. We speculate that a defect in METC may occur primarily by UUO, which then triggers the production of ROS, which then impairs the antioxidative system, and consequently causes the death of interstitial cells and inflammatory reactions. Because we still do not know what directly causes the mitochondrial damage, this issue requires further examination. We also found decreases in the levels of antioxidative enzymes in the kidneys of WT and KO mice with and without AsA (Figure 7). Among the antioxidative enzymes, GPX1 exhibited the most marked decrease, which is consistent with the decreased GPX activity observed in kidneys with UUO [7]. GPX1 is a selenoprotein that contains a selenocysteine (SeCys) residue at the catalytic center. Nitric oxide that is induced by inflammation causes inactivation of GPX1 by oxidizing SeCys [35]. Hydrogen peroxide converts the SeCys to dehydroalanine and results in irreversible inactivation [36]. The conversion from SeCys to dehydroalanine in vivo is indeed demonstrated in the liver of SOD1deficient mice [37]. Although superoxide directly inactivates GPX1 [38], we have reported no changes in the levels of the GPX1 protein between SOD1-deficient and WT mice kidneys [39]. Thus, hydrogen peroxide, but not superoxide, appears to be involved in the pathogenesis of kidneys with UUO. We observed the elevated production of some proteins that reside in the ER lumen (Figure 5C). Accumulation of a large body of damaged proteins in the ER causes ER stress, which ultimately leads to cell death under extreme situations [40]. PRDX4 together with ERO1 appears to be required for collagen production because a deficiency decreases AsA and results in decreased collagen production [41]. However, it is unlikely that ER stress was the cause of renal cell death in the UUO model as follows.
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
Mitochondrial dysfunction in renal failure
We observed that the expression of PRDX4, a protein thiol oxidase responsible for oxidative protein folding in the ER, was markedly induced in the kidneys by UUO, but that the levels of PDI and Bip, other hallmarks of ER stress, were increased only slightly (Figure 5C). Because PRDX4 also was secreted [42], the elevated levels of PRDX4 in the kidneys as a result of UUO may have reflected the presence of extracellular PRDX4. In fact, overexpressed PRDX4 ameliorates liver fibrosis by suppressing oxidative stress [43]. If ER stress were the underlying mechanism for the renal damage, collagen production would conversely be decreased due to the dysfunction of the ER. Thus, the elevation in PRDX4 together with other ER resident proteins appears to have been an adaptive response of myofibroblasts to activate ER function for collagen secretion. In conclusion, it was unexpected that no significant difference was observed in collagen production as a result of UUO, irrespective of the amounts of AsA in the mice, which implies either a low demand for AsA or the presence of another electron donor for collagen production in the kidneys. A marked induction of PRDX4 was observed, which would imply an activated ER function in myofibroblasts for the secretion of a large body of collagen. Mitochondrial damage, which occurred at the initial stage of kidney dysfunction, appeared to be a cause of ROS elevation and a further triggering of cell death and fibrosis.
Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
References [1] Klahr S, Morrissey J. Obstructive nephropathy and renal fibrosis. Am J Physiol Renal Physiol 2002;283:F861–F875. [2] Bascands JL, Schanstra JP. Obstructive nephropathy: insights from genetically engineered animals. Kidney Int 2005;68: 925–937. [3] Schreiner GF, Harris KP, Purkerson ML, Klahr S. Immunological aspects of acute ureteral obstruction: immune cell infiltrate in the kidney. Kidney Int 1988;34:487–493. [4] Cachat F, Lange-Sperandio B, Chang AY, Kiley SC, Thornhill BA, Forbes MS, Chevalier RL. Ureteral obstruction in neonatal mice elicits segment-specific tubular cell responses leading to nephron loss. Kidney Int 2003;63:564–575. [5] Rouschop KM, Claessen N, Pals ST, Weening JJ, Florquin S. CD44 disruption prevents degeneration of the capillary network in obstructive nephropathy via reduction of TGFbeta1-induced apoptosis. J Am Soc Nephrol 2006;17:746–753. [6] Kawada N, Moriyama T, Ando A, Fukunaga M, Miyata T, Kurokawa K, et al. Increased oxidative stress in mouse kidneys with unilateral ureteral obstruction. Kidney Int 1999; 56:1004–1013. [7] Kinter M, Wolstenholme JT, Thornhill BA, Newton EA, McCormick ML, Chevalier RL. Unilateral ureteral obstruction impairs renal antioxidant enzyme activation during sodium depletion. Kidney Int 1999;55:1327–1334.
1123
[8] Sunami R, Sugiyama H, Wang DH, Kobayashi M, Maeshima Y, Yamasaki Y, et al. Acatalasemia sensitizes renal tubular epithelial cells to apoptosis and exacerbates renal fibrosis after unilateral ureteral obstruction. Am J Physiol Renal Physiol 2004;286:F1030–F1038. [9] Dendooven A, Ishola DA Jr, Nguyen TQ, Van der Giezen DM, Kok RJ, Goldschmeding R, Joles JA. Oxidative stress in obstructive nephropathy. Int J Exp Pathol 2011;92:202–210. [10] Fujii J, Ito JI, Zhang X, Kurahashi T. Unveiling the roles of the glutathione redox system in vivo by analyzing genetically modified mice. J Clin Biochem Nutr 2011;49:70–78. [11] Du J, Cullen JJ, Buettner GR. Ascorbic acid: chemistry, biology and the treatment of cancer. Biochim Biophys Acta 2012;1826:443–457. [12] Linster CL, Van Schaftingen E. Vitamin C. Biosynthesis, recycling and degradation in mammals. FEBS J 2007;274: 1–22. [13] Mandl J, Szarka A, Bánhegyi G. Vitamin C: update on physiology and pharmacology. Br J Pharmacol 2009;157: 1097–1110. [14] Deicher R, Hörl WH. Vitamin C in chronic kidney disease and hemodialysis patients. Kidney Blood Press Res 2003; 26:100–106. [15] Coombes JS, Fassett RG. Antioxidant therapy in hemodialysis patients: a systematic review. Kidney Int 2012;81:233–246. [16] Sadat U, Usman A, Gillard JH, Boyle JR. Does ascorbic acid protect against contrast-induced acute kidney injury in patients undergoing coronary angiography: a systematic review with meta-analysis of randomized controlled trials. J Am Coll Cardiol 2013;62;2167–2175. [17] Gabbay KH, Bohren KM, Morello R, Bertin T, Liu J, Vogel P. Ascorbate synthesis pathway: dual role of ascorbate in bone homeostasis. J Biol Chem 2010;285:19510–19520. [18] Takahashi M, Miyata S, Fujii J, Inai Y, Ueyama S, Araki M, et al. In vivo role of aldehyde reductase. Bichim Biophys Acta 2012;1820:1787–1796. [19] Ito J, Otsuki N, Zhang X, Konno K, Kurahashi T, Takahashi M, et al. Ascorbic acid reverses the prolonged anesthetic action of pentobarbital in Akr1a-knockout mice. Life Sci 2014;95:1–8. [20] Takahashi M, Fujii J, Miyoshi E, Hoshi A, Taniguchi N. Elevation of aldose reductase gene expression in rat primary hepatoma and hepatoma cell lines: implication in detoxification of cytotoxic aldehydes. Int J Cancer 1995;62:749–754. [21] Matsuoka Y, Yamato M, Yamasaki T, Mito F, Yamada K. Rapid and convenient detection of ascorbic acid using a fluorescent nitroxide switch. Free Radic Biol Med 2012; 53:2112–2118. [22] Takahashi M, Fujii J, Teshima T, Suzuki K, Shiba T, Taniguchi N. Identity of a major 3-deoxyglucosone-reducing enzyme with aldehyde reductase in rat liver established by amino acid sequencing and cDNA expression. Gene 1993; 127:249–253. [23] Iuchi Y, Okada F, Onuma K, Onoda T, Asao H, Kobayashi M, Fujii J. Elevated oxidative stress in erythrocytes due to an SOD1 deficiency causes anemia and triggers autoantibody production. Biochem J 2007;402:219–227. [24] Ito R, Takahashi M, Ihara H, Tsukamoto H, Fujii J, Ikeda Y. Measurement of peroxiredoxin-4 serum levels in rat tissue and its use as a potential marker for hepatic disease. Mol Med Rep 2012;6:379–384. [25] Fujii T, Endo T, Fujii J, Taniguchi N. Differential expression of glutathione reductase and cytosolic glutathione peroxidase, GPX1, in developing rat lungs and kidneys. Free Radic Res 2002;36:1041–1049. [26] Ishikawa Y, Bächinger HP. A molecular ensemble in the rER for procollagen maturation. Biochim Biophys Acta 2013; 1833:2479–2491. [27] Hinz B, Phan SH, Thannickal VJ, Prunotto M, Desmoulière A, Varga J, et al. Recent developments in myofibroblast biology:
1124 H. Nishida et al.
[28] [29] [30]
[31]
Downloaded by [Karolinska Institutet, University Library] at 16:30 12 September 2015
[32] [33]
[34]
[35]
paradigms for connective tissue remodeling. Am J Pathol 2012;180:1340–1355. Toyokuni S, Mori T, Dizdaroglu M. DNA base modifications in renal chromatin of Wistar rats treated with a renal carcinogen, ferric nitrilotriacetate. Int J Cancer 1994;57:123–128. Parsons KK, Maeda N, Yamauchi M, Banes AJ, Koller BH. Ascorbic acid-independent synthesis of collagen in mice. Am J Physiol Endocrinol Metab 2006;290:E1131–E1139. Forbes MS, Thornhill BA, Chevalier RL. Proximal tubular injury and rapid formation of atubular glomeruli in mice with unilateral ureteral obstruction: a new look at an old model. Am J Physiol Renal Physiol 2011;301:F110–F117. Barnes JL, Gorin Y. Myofibroblast differentiation during fibrosis: role of NAD(P)H oxidases. Kidney Int 2011;79: 944–956. Mailloux RJ, McBride SL, Harper ME. Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics. Trends Biochem Sci 2013;38:592–602. Chung SD, Lai TY, Chien CT, Yu HJ. Activating Nrf-2 signaling depresses unilateral ureteral obstruction-evoked mitochondrial stress-related autophagy, apoptosis and pyroptosis in kidney. PLoS One 2012;7:e47299. Mizuguchi Y, Chen J, Seshan SV, Poppas DP, Szeto HH, Felsen D. A novel cell-permeable antioxidant peptide decreases renal tubular apoptosis and damage in unilateral ureteral obstruction. Am J Physiol Renal Physiol 2008;295:F1545–1553. Asahi M, Fujii J, Takao T, Kuzuya T, Hori M, Shimonishi Y, Taniguchi N. The oxidation of selenocysteine is involved in the inactivation of glutathione peroxidase by nitric oxide donor. J Biol Chem 1997;272:19152–19157.
Supplementary material available online Supplementary Figures 1–3.
[36] Cho CS, Lee S, Lee GT, Woo HA, Choi EJ, Rhee SG. Irreversible inactivation of glutathione peroxidase 1 and reversible inactivation of peroxiredoxin II by H2O2 in red blood cells. Antioxid Redox Signal 2010;12:1235–1246. [37] Wang SK, Weaver JD, Zhang S, Lei XG. Knockout of SOD1 promotes conversion of selenocysteine to dehydroalanine in murine hepatic GPX1 protein. Free Radic Biol Med 2011;51:197–204. [38] Blum J, Fridovich I. Inactivation of glutathione peroxidase by superoxide radical. Arch Biochem Biophys 1985;240: 500–508. [39] Yamanobe T, Okada F, Iuchi Y, Onuma K, Tomita Y, Fujii J. Deterioration of ischemia/reperfusion-induced acute renal failure in SOD1-deficient mice. Free Radic Res 2007;41: 200–207. [40] Walter P, Ron D. The unfolded protein response: from stress pathway to homeostatic regulation. Science 2011;334: 1081–1086. [41] Zito E, Hansen HG, Yeo GS, Fujii J, Ron D. Endoplasmic reticulum thiol oxidase deficiency leads to ascorbic acid depletion and noncanonical scurvy in mice. Mol Cell 2012; 48:39–51. [42] Okado-Matsumoto A, Matsumoto A, Fujii J, Taniguchi N. Peroxiredoxin IV is a secretable protein with heparin-binding properties under reduced conditions. J Biochem 2000;127: 493–501. [43] Nabeshima A, Yamada S, Guo X, Tanimoto A, Wang KY, Shimajiri S, et al. Peroxiredoxin 4 protects against nonalcoholic steatohepatitis and type 2 diabetes in a nongenetic mouse model. Antioxid Redox Signal 2013;19:1983–1998.
