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Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis–trans isomerase activity
BBRC Biochemical and Biophysical Research Communications 316 (2004) 1073–1080 www.elsevier.com/locate/ybbrc
Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis–trans isomerase activity Feng Hong,a Jinhwa Lee,b Yu Ji Piao,a Yeong Kwon Jae,a Young-Joo Kim,a Changkyu Oh,c Jeong-Sun Seo,c Yeon Sook Yun,d Chul Woo Yang,e Joohun Ha,a and Sung Soo Kima,* a
Department of Molecular Biology, Medical Science and Engineering Research Center for Bioreaction to Reactive Oxygen Species, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea b Department of Biotechnology, Dongseo University, Busan 617-716, Republic of Korea c Ilchun Institute for Molecular Medicine, College of Medicine, Seoul National University, Seoul 110-799, Republic of Korea d Laboratory of Immunology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea e Department of Internal Medicine, The Catholic University of Korea, Seoul 137-040, Republic of Korea Received 20 February 2004
Abstract Cyclosporin A (CsA) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug. Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed family member of cyclophilins. We previously showed that CsA toxicity is mediated by ROS generation as well as by inhibition of peptidyl-prolyl cis–trans isomerase (PPIase) activity of CypA in CsA-treated myoblasts [FASEB J. 16 (2002) 1633]. Since CsAinduced nephrotoxicity is the most significant adverse effect in its clinical utilization, we here investigated the role of CsA inhibition of CypA PPIase activity in its nephrotoxicity using transgenic mouse models. Transgenic mice of either wild type (CypA/wt) or R55A PPIase mutant type (CypA/R55A), a dominant negative mutant of CypA PPIase activity, showed normal growth without any apparent abnormalities. However, CsA-induced nephrotoxicity was virtually suppressed in CypA/wt mice, but exacerbated in CypA/ R55A mice, compared to that of littermates. Also, life expectancy was extended in CypA/wt mice and shortened in CypA/R55A mice during CsA administration. Besides, CsA-induced nephrotoxicity was inversely related to the levels of catalase expression and activity. In conclusion, our data provide in vivo evidence that supplement of CypA PPIase activity allows animal’s resistance toward CsA-induced nephrotoxicity. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Cyclosporin A-induced nephrotoxicity; Cyclophilin A; PPIase activity; Catalase
Cyclosporin A (CsA) is a potent and widely used immunosuppressive drug in organ transplantations and autoimmune disorders. However, CsA evokes serious adverse effects that critically limit its clinical utilization [1]. Nephrotoxicity is the most serious and commonly found among CsA-induced adverse effects. Many investigations have attempted to understand the molecular
*
Corresponding author. Fax: +82-2-959-8168. E-mail addresses: [email protected], [email protected] (S.S. Kim). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.160
mechanism(s) of CsA-induced nephrotoxicity and found that reactive oxygen species (ROS) are one of the causative factors for CsA-induced nephrotoxicity [2–4]. Accordingly, co-treatment of CsA with antioxidant drugs such as a-tocopherol [5], lazaroid [6], and N-acetyl-L -cystein [7] has been shown to diminish CsA-induced nephrotoxicity. Also, the possible origins of ROS have been ascribed to cytochrome P450 enzyme system [8], NADPH oxidase [9], xanthine oxidase [10], angiotensin II [11], increased renal nerve activity [12], or involvement of cytokines such as transforming growth factor-b1 [13].
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Cyclophilins are probably found in all the existing organisms and remarkably conserved through evolution. They are named after functioning as an intracellular CsA receptor and suppress immune reactions by inhibiting calcineurin activity after forming complex with CsA [14]. The classical cyclophilins are composed of four isoforms (cyclophilins A, B, C, and D). Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed cyclophilin. The human CypA has a high degree of sequence homology with human cyclophilin B (CypB), C (CypC), and D (CypD). CypB and CypC have the N-terminal sequences which target them to endoplasmic reticulum [15]. CypD is an integral part of the mitochondrial permeability transition complex and plays a crucial role in apoptotic cell death. CypA seems to have dual localization in the cytoplasm and in the nucleus. All family members of the cyclophilins possess enzymatic peptidyl-prolyl cis–trans isomerase (PPIase) activity, the role of which in the protein folding process was first revealed from investigations of collagen folding in vitro. Steinmann et al. [16] demonstrated that CsA blocked the PPIase activity of cyclophilins and significantly delayed maturation of collagen. The role of cyclophilin PPIase activity in protein folding was subsequently confirmed in a similar study using rabbit reticulocyte lysate [17]. In addition to protein folding, the PPIase activity of cyclophilins has recently been suggested to play important roles in diverse cellular processes including intracellular trafficking [18], signal transduction [19], cell cycle regulation [20], transcription regulation [21], differentiation [22], and maintenance of multi-protein complex stability [14,23]. CypA has recently been recognized for the protective role, especially functioning as an antioxidant [24]. Moreover, introduction of wild type CypA (CypA/wt) into SOD mutant cells increases cell viability [25], while introduction of point mutant R55A (CypA/R55A) that functions as a dominant negative mutant of PPIase activity [26] decreases cell viability [25]. We have also shown the dose-dependent cytoprotection along with anti-ROS action of CypA PPIase using H9c2 cardiac myoblasts. In addition, we have demonstrated that the induced expression of CypA on the treatment of low CsA concentration provides cells with an acquired tolerance to higher CsA concentrations [27]. In this report, we investigated if CsA evokes nephrotoxicity by inhibiting CypA PPIase activity and if overexpression of CypA/wt can protect against CsA-induced nephrotoxicity in vivo, using transgenic mice overexpressing CypA/ wt and CypA/R55A.
Materials and methods Generation of transgenic mice. Plasmids containing CypA/wt and CypA/R55A transgene were described previously [27]. The vectors
linearized with NruI were injected into the pronucleus of fertilized single cell eggs of FVB mice (Jackson laboratory, Bar Harbor, ME). These products were then surgically transferred into the oviduct of pseudopregnant female mice. Germline transmission was confirmed as follows. After constitution of transgenic mice, chromosomal DNA was extracted from mice tails and used as templates for PCR. CypA/wt and CypA/R55A transgene fragments were recovered at 630 base pair size using primers (50 -GGCACCAAAATCAACGGG-30 ; 50 -GCGGATCC GAGTTGTCCACAGTCGGA-30 ) by PCR analysis. Animal experimental protocol. CsA-induced nephropathies were made with minor modifications of previous reports as follows [28–30]. Mice weighing 20–25 g were subjected to a low-salt diet (0.01% sodium, Teklad Premier, Madison, WI). Control group (n ¼ 12) received vehicle (olive oil) alone intraperitoneally (IP). Experimental groups (12 mice for each group) received 80 or 50 mg/kg CsA IP daily for short- or long-term treatment, respectively. Animals were sacrificed for shortterm treatment at 7 (littermate and CypA/wt groups) or 4 days (CypA/ R55A mice) and for long-term treatment at 28 (littermate and CypA/ wt groups) or 14 days (CypA/R55A mice) to get blood and tissue samplings. Because of high toxicity and early death, CypA/R55A mice were processed earlier. Blood withdrawn from the ophthalmic vein on the final day was used for the measurement of blood urea nitrogen (BUN) and creatinine using a standard kit (Sigma Chemical, St. Louis, MO). After blood sampling, kidneys were rapidly removed, fixed in formaldehyde, cut into 4-lm sections, and stained with hematoxylin– eosin (HE) for histological analysis. Catalase assay. Catalase activity was measured by using AMPLEX RED CATALASE ASSAY KIT (A-22180) (Molecular Probes, Eugene, OR) according to the manufacturer’s protocol. Briefly, tissue homogenates (3 lg/25 ll) were incubated with 25 ll of 40 lM H2 O2 for 30 min at room temperature. Thereafter, 50 ll of the Amplex Red/HRP working solution was added and incubated for 30 min at 37 °C. Fluorescence was measured using excitation at 560 nm and emission at 590 nm with a fluorescence microplate reader (HTS 7000; Perkin– Elmer). Superoxide dismutase assay. CuZn- and Mn-SOD activities were measured by using BIOXYTECH SOD-525 kit (OxisResearch, Portland, OR) according to the manufacturer’s protocol. To prepare extracts specific for CuZn-SOD, ethanol–chloroform extraction was used for inactivating Mn-SOD. Tissue homogenates (10 lg/40 ll) were incubated with 900 ll assay buffer and 30 ll of 1,4,6-trimethyl-2-vinylpyridinium-trifluoromethanesulfonate solution for 1 min at 37 °C. Thereafter, 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c] fluorene solution (30 ll) was added and the optical densities of 525 nm absorbance were read. For standard curve generation, a red blood cell lysate was prepared. Serial fold dilution of the lysate yielded a good correlation of SOD activity (SOD activity ¼ 0.123X + 0.0354, where X ¼ dilution factor of the lysate; data not shown). All reactions were performed at room temperature. Glutathione peroxidase assay. Glutathione peroxidase activity was measured using BIOXYTECH GPx-340 kit (OxisResearch, Portland, OR) according to the manufacturer’s protocol. Renal tissues were homogenized in 500 ll lysis buffer consisting of 50 mM Tris–HCl, pH 7.5, 5 mM EDTA, and 1 mM of 2-mercaptoethanol. Assay buffer (350 ll), NADPH reagent (350 ll), and tissue homogenates (10 lg/70 ll) were put into a cuvette and placed in the spectrophotometer. Thereafter, 350 ll of tert-butyl hydroperoxide was added. Optical density of 340 nm was monitored at every 30 s for 3 min. For standard curve generation, BIOXYTECH Cellular Glutathione Peroxidase Control was used and a good correlation was observed between known amounts of the glutathione peroxidase control and enzyme activity estimated by NADPH consumption (P < 0:0001, data not shown). All reactions were performed at room temperature. Western blot analysis. Total proteins (30 lg) from tissue homogenates were separated by SDS–PAGE and transferred onto nitrocellulose membrane. After blocking, the membrane was incubated
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with the indicated primary antibody followed by incubation with a secondary antibody. ECL-plus chemiluminescence (Amersham–Pharmacia Biotech, Piscataway, NJ) and X-ray film exposure for detection, and Bio-Rad imaging densitometric scan for quantitation were employed. Antibodies specific to c-Myc and b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), CypA antibody was from Upstate Biotechnology (Lake Placid, NY), and catalase, CuZn-SOD, and Mn-SOD antibodies were from Calbiochem (San Diego, CA). Northern blot analysis. Total RNA was prepared from renal tissues using TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA (15 lg) was loaded per lane on a 1% agarose/formaldehyde gel and transferred onto a nylon membrane (Schleicher & Schuell, Dassel, Germany). Membrane was incubated with probes labeled with [a-32 P]dCTP using Ladderman Labeling Kit (TAKARA Bio, Shiga, Japan) using RT-PCR product as a template. Following several washes, the radioactive signal was detected by exposure to X-ray films. Primers were as follows: catalase, 50 -CCAGGTTTCCTTGTTACGTG-30 and 50 -GTCAAATGCCA TCTGTTGAACCT-30 ; CuZn-SOD, 50 -AGCATGGCGATGAAGGC CGT-30 and 50 -TCATCTTGTTTCTCGTGGACC-30 ; Mn-SOD, 50 -AC CACGCGACCTACGTGAAC-30 and 50 -CCCACACATCAATCCCC AGC-30 ; Gpx, 50 -AGCACCATGTGCGCGGCGCG-30 and 50 -TCCG CATTGCTCCCTGGCTCCT-30 ; and GAPDH, 50 -TGGCAAAGTGG ATATTGTCG-30 and 50 -AGGAGCAGAGATGATGACCC-30 . PCR products were verified by restriction enzyme digestion. Data analysis. Results were expressed as means SE from at least three independent experiments. Statistical analysis was performed using one-way ANOVA tests with PC version of SPSS statistical package (v.11.0). Kaplan–Meier survival curves were statistically assessed by log-rank test. Unless otherwise indicated, P < 0:05 was deemed significant.
Results Generation of transgenic mice overexpressing CypA wild type and R55A mutant type To study in vivo role of CypA or its PPIase activity on CsA toxicity, we generated CypA/wt or CypA/R55A transgenic mice as described in Materials and methods. For each of the CypA/wt and CypA/R55A groups, three founders were generated and germline transmission of both transgenes was confirmed by PCR analysis using chromosomal DNA extracted from mice tails (Fig. 1A). The transgene expression in kidneys of experimental animals was confirmed by Western blot analysis using antibodies against c-Myc or CypA (Fig. 1B). All the transgenes were expressed at levels 1- to 3-fold higher than that of the endogenous CypA (Fig. 1B). Each group of transgenic mice overexpressing CypA/wt or CypA/R55A showed normal growth (Fig. 2) without any gross-anatomical or histological alterations as well as functional abnormalities (data not shown) during the period of growth. PPIase activity is required for the protective effect of CypA on CsA-induced nephrotoxicity in vivo We investigated the protective role of CypA PPIase activity using the constructed transgenic mice. Nephro-
Fig. 1. Construction of CypA/wt and CypA/R55A transgenic mice. (A) Recovery of transgene fragments by PCR is shown to confirm germline transmission of CypA/wt and CypA/R55A positive transgenic mice (+). The littermates ()) are shown as negative controls. (B) Transgene expression in kidney tissues. Protein samples from kidney of each transgenic mouse were subjected to immunoblot using antibodies against c-Myc, CypA or b-actin. b-Actin was used as a loading control. All experiments were done three times and the typical figures are shown. *; **P < 0:01, compared with littermates ()) of CypA/wt or CypA/R55A transgenic mice, respectively.
toxicity was induced by short- and long-term treatments with CsA. To detect nephropathy, we first measured blood urea nitrogen (BUN) and creatinine levels. CsA at 80 or 50 mg/kg concentration for both short- and longterm administration, respectively, showed the increased BUN and creatinine levels in littermates. In the CypA/ wt group, however, no significant increase in BUN or creatinine level was observed under administration of the same dose of CsA. In contrast, the BUN and creatinine increases in both the short- and long-term CsA treatments in CypA/R55A group were several fold amplified (Figs. 3A and B). Histological examination was next performed using hematoxylin–eosin staining method. CypA/wt transgenic mice did not manifest any lesions or damage in kidney architecture both in shortand long-term treatments (Fig. 4). In contrast, focal lesions with small infiltrates of acute inflammatory cells were evident in the littermate control and substantially larger in the CypA/R55A group after short-term
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Fig. 2. Growth curves of transgenic mice. Body weights of male (#, open symbols) and female ($, filled symbols) transgenic mice as well as littermates were measured at the designated days during 4–8 weeks after birth. Data are means (n ¼ 6) SE.
treatment. Despite the clear signs of damage, the typical histological alterations including cytoplasmic microvacuolization of the tubular epithelial cells and heavy infiltration of acute inflammatory cells that were histological hallmarks of acute CsA-induced nephrotoxicity were not observed in our short-term specimen. Similarly, the long-term treatment induced microvacuolization in the littermate group and more abundantly in the CypA/R55A, though this change was atypical rather than the classical signs of chronic CsA-induced nephrotoxicity that included tubulointerstitial fibrosis, tubular atrophy, and glomerulosclerosis. Since mouse model for CsA-induced nephrotoxicity, in contrast to rat model, is yet to be established, atypical histological changes in our samples might be understandable. Nonetheless, both biochemical and microscopical examinations unambiguously displayed that overexpression of CypA/wt suppressed the CsA-induced nephropathy, but overexpression of CypA/R55A exaggerated the damage in both short- and long-term treatments. Correspondingly, the CypA/wt mice survived longer than the littermate, but the CypA/R55A mice died much earlier during short- and long-term administration of CsA (Figs. 5A and B). In fact, CypA/wt mice were maintained normal without any increase in BUN and creatinine levels even 30 and 60 days after initiation of CsA daily administration at 80 or 50 mg/kg concentration, respectively. In contrast, most of littermates died by these times (data not shown). Although we presented the above data obtained in mice expressing CypA/wt or CypA/ R55A most highly, we also observed that the expression level of the transgene, CypA/wt or CypA/R55A, determined the severity of CsA-induced nephropathy in a dose-dependent manner (data not shown).
Fig. 3. Effect of CypA/wt or CypA/R55A overexpression on CsA-induced nephrotoxicity in mice. BUN (A) and creatinine (B) levels were determined using samples prepared as described in Materials and methods. Data represent means (n ¼ 12) SE. *; **P < 0:05, compared with littermate without CsA administration for short- or longterm experiment, respectively.
Effect of CypA PPIase activity on antioxidant enzymes Given that CsA treatment impairs different antioxidant enzymes in different experimental conditions [31,32], we investigated the effect of CypA on the antioxidant enzymes under our experimental conditions. CypA/wt or CypA/R55A overexpression by itself did not cause any changes in antioxidant enzymes at both mRNA and protein levels (Fig. 6). However, CsA treatment reduced the catalase expression and activity levels in littermates and further in CypA/R55A transgenic mice. In contrast, it increased the catalase expression and activity levels in CypA/wt transgenic mice (Figs. 6 and 7). Interestingly, the expression and activity levels of other antioxidant enzymes including CuZnSOD, Mn-SOD, and glutathione peroxidase did not change at all in all experimental animals even after CsA
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Fig. 4. Histological changes in kidney sections after CsA administration. Kidney samples were cut after fixation with formaldehyde and stained with HE for histological examinations. Typical pictures are presented to show CypA PPIase effect on CsA-induced nephrotoxicity. Rectangular areas (100) are enlarged to show typical features in detail (400). Arrowheads specifically indicate pathological changes.
Fig. 5. Kaplan–Meier survival curves of experimental mice during CsA treatment. Numbers of surviving CypA/wt (open circle) or CypA/ R55A (closed circle) transgenic mice or littermates (triangle) during CsA treatment were plotted for 7 or 28 days after CsA administration for short- or long-term experiment, respectively (P < 0:05).
Fig. 6. Effect of CypA/wt or CypA/R55A overexpression on expression of antioxidant enzymes in mice. Samples for Western (A) and Northern blot analyses (B) were prepared from kidneys of short- and long-term CsA-treated littermate, CypA/wt or CypA/R55A transgenic mice. Typical figures were demonstrated from three independent experiments. b-Actin was used as a loading control for Western blot. GAPDH transcript was used for RNA loading control.
Discussion treatment. Again, expression levels of transgenes in CypA/wt or CypA/R55A transgenic mouse lines appeared to determine the expression and activity levels of catalase after CsA treatment as observed in CsA-induced nephropathy (data not shown).
In this report, we first demonstrated in vivo that overexpression of CypA/wt can eliminate CsA-induced nephrotoxicity via CypA PPIase activity. The conclusion was evidenced by our observations that transgenic
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Fig. 7. Effect of CypA/wt or CypA/R55A overexpression on antioxidant enzyme activities in mice. Activity assays of catalase (A), CuZn-SOD (B), Mn-SOD (C), and GPx (D) from kidney samples of transgenic mice after CsA administration were performed as described in Materials and methods. Data represent means SE of three independent experiments. *; **P < 0:05, compared with littermate without CsA administration for short- or long-term experiment, respectively.
mice overexpressing CypA/wt were more resistant to CsA-induced nephrotoxicity, but those overexpressing CypA/R55A, a dominant negative mutant type of CypA PPIase activity, were more susceptible than littermates. In addition, the expression level of the transgenes, both CypA/wt and CypA/R55A, affected severity of CsA-induced nephrotoxicity. One possible explanation is that CsA titrates out CypA PPIase activity by forming a CsA–CypA complex when magnitude of PPIase activity determines the protective capacity against CsA. We have established the dosage-dependent protective effect of CypA PPIase on CsA-induced cytotoxicity more thoroughly in CypA/wt or CypA/R55A transfectants of MDCK renal tubular epithelial cells by observing that the ectopic expression level of either gene was directly proportional to the relieving or exacerbating capacity over the CsA-induced cytotoxicity (data not shown). ROS generation has been attributed to CsA-induced toxicity including nephrotoxicity [2–4]. Our finding that CypA/wt and CypA/R55A transfectants in MDCK cells (data not shown) as well as in H9c2 cardiac myoblasts [27] are more resistant or susceptible to H2 O2 , respectively, implies that CypA PPIase may function as an antioxidant. The anti-ROS function of CypA PPIase
activity derives an idea that cellular defense machinery against ROS might be a direct or indirect target of cis– trans isomerization. Also available are reports that CsA changes expression levels of different antioxidant enzymes [31,32]. To map out the target molecules, we determined alterations in the expression level and activity of antioxidant enzymes in our experimental animals in the absence or presence of CsA. Upon CsA treatment, catalase, but no other antioxidant enzymes, showed a dual regulatory pattern although absence of CsA negated the regulatory changes among littermate, CypA/ wt, and CypA/R55A transgenic mice: CsA down-regulated catalase expression in littermates, further in CypA/ R55A transgenic mice, but up-regulated it in CypA/wt transgenic mice. These data clearly exclude the possibility that catalase is a direct target of CypA PPIase activity. Simple explanation would be that mild oxidative stress positively regulates catalase expression but death decision by severe oxidative stress negatively regulates it. This can be supported by a recent report that low oxidative stress activates catalase expression through its interaction with c-Abl and Arg, while high level of ROS that evokes apoptosis has an antagonistic effect on catalase expression [33]. Indeed, our study in
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MDCK cells also displayed the dual regulatory mode between ROS generation and catalase expression. CsA and H2 O2 increased catalase expression in MDCK cells within the concentrations that cells could sustain, but decreased it beyond those concentrations in a dose-dependent manner (data not shown). Therefore, we surmise that catalase expression is regulated secondarily by intracellular ROS level rather than directly by CypA PPIase activity. At the present time, how CypA PPIase activity scavenges ROS remains to be elucidated. Inducibility of endogenous CypA has been reported under mild oxidative stress [34]. We also showed in the previous report that low CsA concentration induced endogenous CypA expression through generation of mild oxidative stress and the induced CypA provided resistance to higher CsA concentrations in H9c2 cardiac myoblasts [27]. MDCK cells showed the same effect of the CsA-CypA induction and resistance as H9c2 cells (data not shown). Interestingly, CypA induction has been reported in papillomavirus-immortalized oral keratinocytes and non-small cell lung cancer [35,36]. In addition, there are accumulating evidences that CypA expression is induced in diverse cells and tissues under different conditions, though the role of increased CypA expression has not been explored. CypA expression increases in mesenchymal stem cells [37], ultraviolet-radiated HeLa cells [38], and thrombinactivated platelets [39]. Importantly, our data show that overexpression of CypA is undisruptive as well as nontoxic to organ integrity including kidney in transgenic mice, excluding the possibility that overexpression of CypA is oncogenic or toxic to cell survival. Rather, it seems that CypA overexpression provides cells with more resistance to hypoxic or stress condition. As prevention of CsA toxicity is highly required in the clinics, our data may provide grounds for searching agents that induce CypA expression to relieve CsA nephrotoxicity. In conclusion, we suggest that CsA inhibition of CypA PPIase activity is a possible mechanism of CsAinduced nephrotoxicity. Besides, we showed that CypA overexpression is not only harmless in experimental animals but also protective against CsA-induced nephrotoxicity. To understand the molecular mechanism(s) of CypA PPIase as an antioxidant, we are currently trying to identify substrates of CypA PPIase. Acknowledgments We are grateful to Dr. Eric N. Olson at University of Texas Southwestern Medical Center for the critical reading of the manuscript and helpful discussion. We also thank Dr. Youn-Wha Kim at Kyung Hee University for technical assistance in histological analysis. This work was funded by grants from the Korea Science and Engineering Foundation (No. R13-2002-020-01001-0) and the Ministry of Public Health and Welfare (No. 03-PJ10-PG6-E001-0001), Republic of Korea.
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Transgenic mice overexpressing cyclophilin A are resistant to cyclosporin A-induced nephrotoxicity via peptidyl-prolyl cis–trans isomerase activity Feng Hong,a Jinhwa Lee,b Yu Ji Piao,a Yeong Kwon Jae,a Young-Joo Kim,a Changkyu Oh,c Jeong-Sun Seo,c Yeon Sook Yun,d Chul Woo Yang,e Joohun Ha,a and Sung Soo Kima,* a
Department of Molecular Biology, Medical Science and Engineering Research Center for Bioreaction to Reactive Oxygen Species, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea b Department of Biotechnology, Dongseo University, Busan 617-716, Republic of Korea c Ilchun Institute for Molecular Medicine, College of Medicine, Seoul National University, Seoul 110-799, Republic of Korea d Laboratory of Immunology, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea e Department of Internal Medicine, The Catholic University of Korea, Seoul 137-040, Republic of Korea Received 20 February 2004
Abstract Cyclosporin A (CsA) suppresses immune reaction by inhibiting calcineurin activity after forming complex with cyclophilins and is currently widely used as an immunosuppressive drug. Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed family member of cyclophilins. We previously showed that CsA toxicity is mediated by ROS generation as well as by inhibition of peptidyl-prolyl cis–trans isomerase (PPIase) activity of CypA in CsA-treated myoblasts [FASEB J. 16 (2002) 1633]. Since CsAinduced nephrotoxicity is the most significant adverse effect in its clinical utilization, we here investigated the role of CsA inhibition of CypA PPIase activity in its nephrotoxicity using transgenic mouse models. Transgenic mice of either wild type (CypA/wt) or R55A PPIase mutant type (CypA/R55A), a dominant negative mutant of CypA PPIase activity, showed normal growth without any apparent abnormalities. However, CsA-induced nephrotoxicity was virtually suppressed in CypA/wt mice, but exacerbated in CypA/ R55A mice, compared to that of littermates. Also, life expectancy was extended in CypA/wt mice and shortened in CypA/R55A mice during CsA administration. Besides, CsA-induced nephrotoxicity was inversely related to the levels of catalase expression and activity. In conclusion, our data provide in vivo evidence that supplement of CypA PPIase activity allows animal’s resistance toward CsA-induced nephrotoxicity. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Cyclosporin A-induced nephrotoxicity; Cyclophilin A; PPIase activity; Catalase
Cyclosporin A (CsA) is a potent and widely used immunosuppressive drug in organ transplantations and autoimmune disorders. However, CsA evokes serious adverse effects that critically limit its clinical utilization [1]. Nephrotoxicity is the most serious and commonly found among CsA-induced adverse effects. Many investigations have attempted to understand the molecular
*
Corresponding author. Fax: +82-2-959-8168. E-mail addresses: [email protected], [email protected] (S.S. Kim). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.160
mechanism(s) of CsA-induced nephrotoxicity and found that reactive oxygen species (ROS) are one of the causative factors for CsA-induced nephrotoxicity [2–4]. Accordingly, co-treatment of CsA with antioxidant drugs such as a-tocopherol [5], lazaroid [6], and N-acetyl-L -cystein [7] has been shown to diminish CsA-induced nephrotoxicity. Also, the possible origins of ROS have been ascribed to cytochrome P450 enzyme system [8], NADPH oxidase [9], xanthine oxidase [10], angiotensin II [11], increased renal nerve activity [12], or involvement of cytokines such as transforming growth factor-b1 [13].
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Cyclophilins are probably found in all the existing organisms and remarkably conserved through evolution. They are named after functioning as an intracellular CsA receptor and suppress immune reactions by inhibiting calcineurin activity after forming complex with CsA [14]. The classical cyclophilins are composed of four isoforms (cyclophilins A, B, C, and D). Cyclophilin A (CypA) is the most abundantly and ubiquitously expressed cyclophilin. The human CypA has a high degree of sequence homology with human cyclophilin B (CypB), C (CypC), and D (CypD). CypB and CypC have the N-terminal sequences which target them to endoplasmic reticulum [15]. CypD is an integral part of the mitochondrial permeability transition complex and plays a crucial role in apoptotic cell death. CypA seems to have dual localization in the cytoplasm and in the nucleus. All family members of the cyclophilins possess enzymatic peptidyl-prolyl cis–trans isomerase (PPIase) activity, the role of which in the protein folding process was first revealed from investigations of collagen folding in vitro. Steinmann et al. [16] demonstrated that CsA blocked the PPIase activity of cyclophilins and significantly delayed maturation of collagen. The role of cyclophilin PPIase activity in protein folding was subsequently confirmed in a similar study using rabbit reticulocyte lysate [17]. In addition to protein folding, the PPIase activity of cyclophilins has recently been suggested to play important roles in diverse cellular processes including intracellular trafficking [18], signal transduction [19], cell cycle regulation [20], transcription regulation [21], differentiation [22], and maintenance of multi-protein complex stability [14,23]. CypA has recently been recognized for the protective role, especially functioning as an antioxidant [24]. Moreover, introduction of wild type CypA (CypA/wt) into SOD mutant cells increases cell viability [25], while introduction of point mutant R55A (CypA/R55A) that functions as a dominant negative mutant of PPIase activity [26] decreases cell viability [25]. We have also shown the dose-dependent cytoprotection along with anti-ROS action of CypA PPIase using H9c2 cardiac myoblasts. In addition, we have demonstrated that the induced expression of CypA on the treatment of low CsA concentration provides cells with an acquired tolerance to higher CsA concentrations [27]. In this report, we investigated if CsA evokes nephrotoxicity by inhibiting CypA PPIase activity and if overexpression of CypA/wt can protect against CsA-induced nephrotoxicity in vivo, using transgenic mice overexpressing CypA/ wt and CypA/R55A.
Materials and methods Generation of transgenic mice. Plasmids containing CypA/wt and CypA/R55A transgene were described previously [27]. The vectors
linearized with NruI were injected into the pronucleus of fertilized single cell eggs of FVB mice (Jackson laboratory, Bar Harbor, ME). These products were then surgically transferred into the oviduct of pseudopregnant female mice. Germline transmission was confirmed as follows. After constitution of transgenic mice, chromosomal DNA was extracted from mice tails and used as templates for PCR. CypA/wt and CypA/R55A transgene fragments were recovered at 630 base pair size using primers (50 -GGCACCAAAATCAACGGG-30 ; 50 -GCGGATCC GAGTTGTCCACAGTCGGA-30 ) by PCR analysis. Animal experimental protocol. CsA-induced nephropathies were made with minor modifications of previous reports as follows [28–30]. Mice weighing 20–25 g were subjected to a low-salt diet (0.01% sodium, Teklad Premier, Madison, WI). Control group (n ¼ 12) received vehicle (olive oil) alone intraperitoneally (IP). Experimental groups (12 mice for each group) received 80 or 50 mg/kg CsA IP daily for short- or long-term treatment, respectively. Animals were sacrificed for shortterm treatment at 7 (littermate and CypA/wt groups) or 4 days (CypA/ R55A mice) and for long-term treatment at 28 (littermate and CypA/ wt groups) or 14 days (CypA/R55A mice) to get blood and tissue samplings. Because of high toxicity and early death, CypA/R55A mice were processed earlier. Blood withdrawn from the ophthalmic vein on the final day was used for the measurement of blood urea nitrogen (BUN) and creatinine using a standard kit (Sigma Chemical, St. Louis, MO). After blood sampling, kidneys were rapidly removed, fixed in formaldehyde, cut into 4-lm sections, and stained with hematoxylin– eosin (HE) for histological analysis. Catalase assay. Catalase activity was measured by using AMPLEX RED CATALASE ASSAY KIT (A-22180) (Molecular Probes, Eugene, OR) according to the manufacturer’s protocol. Briefly, tissue homogenates (3 lg/25 ll) were incubated with 25 ll of 40 lM H2 O2 for 30 min at room temperature. Thereafter, 50 ll of the Amplex Red/HRP working solution was added and incubated for 30 min at 37 °C. Fluorescence was measured using excitation at 560 nm and emission at 590 nm with a fluorescence microplate reader (HTS 7000; Perkin– Elmer). Superoxide dismutase assay. CuZn- and Mn-SOD activities were measured by using BIOXYTECH SOD-525 kit (OxisResearch, Portland, OR) according to the manufacturer’s protocol. To prepare extracts specific for CuZn-SOD, ethanol–chloroform extraction was used for inactivating Mn-SOD. Tissue homogenates (10 lg/40 ll) were incubated with 900 ll assay buffer and 30 ll of 1,4,6-trimethyl-2-vinylpyridinium-trifluoromethanesulfonate solution for 1 min at 37 °C. Thereafter, 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c] fluorene solution (30 ll) was added and the optical densities of 525 nm absorbance were read. For standard curve generation, a red blood cell lysate was prepared. Serial fold dilution of the lysate yielded a good correlation of SOD activity (SOD activity ¼ 0.123X + 0.0354, where X ¼ dilution factor of the lysate; data not shown). All reactions were performed at room temperature. Glutathione peroxidase assay. Glutathione peroxidase activity was measured using BIOXYTECH GPx-340 kit (OxisResearch, Portland, OR) according to the manufacturer’s protocol. Renal tissues were homogenized in 500 ll lysis buffer consisting of 50 mM Tris–HCl, pH 7.5, 5 mM EDTA, and 1 mM of 2-mercaptoethanol. Assay buffer (350 ll), NADPH reagent (350 ll), and tissue homogenates (10 lg/70 ll) were put into a cuvette and placed in the spectrophotometer. Thereafter, 350 ll of tert-butyl hydroperoxide was added. Optical density of 340 nm was monitored at every 30 s for 3 min. For standard curve generation, BIOXYTECH Cellular Glutathione Peroxidase Control was used and a good correlation was observed between known amounts of the glutathione peroxidase control and enzyme activity estimated by NADPH consumption (P < 0:0001, data not shown). All reactions were performed at room temperature. Western blot analysis. Total proteins (30 lg) from tissue homogenates were separated by SDS–PAGE and transferred onto nitrocellulose membrane. After blocking, the membrane was incubated
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with the indicated primary antibody followed by incubation with a secondary antibody. ECL-plus chemiluminescence (Amersham–Pharmacia Biotech, Piscataway, NJ) and X-ray film exposure for detection, and Bio-Rad imaging densitometric scan for quantitation were employed. Antibodies specific to c-Myc and b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), CypA antibody was from Upstate Biotechnology (Lake Placid, NY), and catalase, CuZn-SOD, and Mn-SOD antibodies were from Calbiochem (San Diego, CA). Northern blot analysis. Total RNA was prepared from renal tissues using TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA (15 lg) was loaded per lane on a 1% agarose/formaldehyde gel and transferred onto a nylon membrane (Schleicher & Schuell, Dassel, Germany). Membrane was incubated with probes labeled with [a-32 P]dCTP using Ladderman Labeling Kit (TAKARA Bio, Shiga, Japan) using RT-PCR product as a template. Following several washes, the radioactive signal was detected by exposure to X-ray films. Primers were as follows: catalase, 50 -CCAGGTTTCCTTGTTACGTG-30 and 50 -GTCAAATGCCA TCTGTTGAACCT-30 ; CuZn-SOD, 50 -AGCATGGCGATGAAGGC CGT-30 and 50 -TCATCTTGTTTCTCGTGGACC-30 ; Mn-SOD, 50 -AC CACGCGACCTACGTGAAC-30 and 50 -CCCACACATCAATCCCC AGC-30 ; Gpx, 50 -AGCACCATGTGCGCGGCGCG-30 and 50 -TCCG CATTGCTCCCTGGCTCCT-30 ; and GAPDH, 50 -TGGCAAAGTGG ATATTGTCG-30 and 50 -AGGAGCAGAGATGATGACCC-30 . PCR products were verified by restriction enzyme digestion. Data analysis. Results were expressed as means SE from at least three independent experiments. Statistical analysis was performed using one-way ANOVA tests with PC version of SPSS statistical package (v.11.0). Kaplan–Meier survival curves were statistically assessed by log-rank test. Unless otherwise indicated, P < 0:05 was deemed significant.
Results Generation of transgenic mice overexpressing CypA wild type and R55A mutant type To study in vivo role of CypA or its PPIase activity on CsA toxicity, we generated CypA/wt or CypA/R55A transgenic mice as described in Materials and methods. For each of the CypA/wt and CypA/R55A groups, three founders were generated and germline transmission of both transgenes was confirmed by PCR analysis using chromosomal DNA extracted from mice tails (Fig. 1A). The transgene expression in kidneys of experimental animals was confirmed by Western blot analysis using antibodies against c-Myc or CypA (Fig. 1B). All the transgenes were expressed at levels 1- to 3-fold higher than that of the endogenous CypA (Fig. 1B). Each group of transgenic mice overexpressing CypA/wt or CypA/R55A showed normal growth (Fig. 2) without any gross-anatomical or histological alterations as well as functional abnormalities (data not shown) during the period of growth. PPIase activity is required for the protective effect of CypA on CsA-induced nephrotoxicity in vivo We investigated the protective role of CypA PPIase activity using the constructed transgenic mice. Nephro-
Fig. 1. Construction of CypA/wt and CypA/R55A transgenic mice. (A) Recovery of transgene fragments by PCR is shown to confirm germline transmission of CypA/wt and CypA/R55A positive transgenic mice (+). The littermates ()) are shown as negative controls. (B) Transgene expression in kidney tissues. Protein samples from kidney of each transgenic mouse were subjected to immunoblot using antibodies against c-Myc, CypA or b-actin. b-Actin was used as a loading control. All experiments were done three times and the typical figures are shown. *; **P < 0:01, compared with littermates ()) of CypA/wt or CypA/R55A transgenic mice, respectively.
toxicity was induced by short- and long-term treatments with CsA. To detect nephropathy, we first measured blood urea nitrogen (BUN) and creatinine levels. CsA at 80 or 50 mg/kg concentration for both short- and longterm administration, respectively, showed the increased BUN and creatinine levels in littermates. In the CypA/ wt group, however, no significant increase in BUN or creatinine level was observed under administration of the same dose of CsA. In contrast, the BUN and creatinine increases in both the short- and long-term CsA treatments in CypA/R55A group were several fold amplified (Figs. 3A and B). Histological examination was next performed using hematoxylin–eosin staining method. CypA/wt transgenic mice did not manifest any lesions or damage in kidney architecture both in shortand long-term treatments (Fig. 4). In contrast, focal lesions with small infiltrates of acute inflammatory cells were evident in the littermate control and substantially larger in the CypA/R55A group after short-term
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Fig. 2. Growth curves of transgenic mice. Body weights of male (#, open symbols) and female ($, filled symbols) transgenic mice as well as littermates were measured at the designated days during 4–8 weeks after birth. Data are means (n ¼ 6) SE.
treatment. Despite the clear signs of damage, the typical histological alterations including cytoplasmic microvacuolization of the tubular epithelial cells and heavy infiltration of acute inflammatory cells that were histological hallmarks of acute CsA-induced nephrotoxicity were not observed in our short-term specimen. Similarly, the long-term treatment induced microvacuolization in the littermate group and more abundantly in the CypA/R55A, though this change was atypical rather than the classical signs of chronic CsA-induced nephrotoxicity that included tubulointerstitial fibrosis, tubular atrophy, and glomerulosclerosis. Since mouse model for CsA-induced nephrotoxicity, in contrast to rat model, is yet to be established, atypical histological changes in our samples might be understandable. Nonetheless, both biochemical and microscopical examinations unambiguously displayed that overexpression of CypA/wt suppressed the CsA-induced nephropathy, but overexpression of CypA/R55A exaggerated the damage in both short- and long-term treatments. Correspondingly, the CypA/wt mice survived longer than the littermate, but the CypA/R55A mice died much earlier during short- and long-term administration of CsA (Figs. 5A and B). In fact, CypA/wt mice were maintained normal without any increase in BUN and creatinine levels even 30 and 60 days after initiation of CsA daily administration at 80 or 50 mg/kg concentration, respectively. In contrast, most of littermates died by these times (data not shown). Although we presented the above data obtained in mice expressing CypA/wt or CypA/ R55A most highly, we also observed that the expression level of the transgene, CypA/wt or CypA/R55A, determined the severity of CsA-induced nephropathy in a dose-dependent manner (data not shown).
Fig. 3. Effect of CypA/wt or CypA/R55A overexpression on CsA-induced nephrotoxicity in mice. BUN (A) and creatinine (B) levels were determined using samples prepared as described in Materials and methods. Data represent means (n ¼ 12) SE. *; **P < 0:05, compared with littermate without CsA administration for short- or longterm experiment, respectively.
Effect of CypA PPIase activity on antioxidant enzymes Given that CsA treatment impairs different antioxidant enzymes in different experimental conditions [31,32], we investigated the effect of CypA on the antioxidant enzymes under our experimental conditions. CypA/wt or CypA/R55A overexpression by itself did not cause any changes in antioxidant enzymes at both mRNA and protein levels (Fig. 6). However, CsA treatment reduced the catalase expression and activity levels in littermates and further in CypA/R55A transgenic mice. In contrast, it increased the catalase expression and activity levels in CypA/wt transgenic mice (Figs. 6 and 7). Interestingly, the expression and activity levels of other antioxidant enzymes including CuZnSOD, Mn-SOD, and glutathione peroxidase did not change at all in all experimental animals even after CsA
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Fig. 4. Histological changes in kidney sections after CsA administration. Kidney samples were cut after fixation with formaldehyde and stained with HE for histological examinations. Typical pictures are presented to show CypA PPIase effect on CsA-induced nephrotoxicity. Rectangular areas (100) are enlarged to show typical features in detail (400). Arrowheads specifically indicate pathological changes.
Fig. 5. Kaplan–Meier survival curves of experimental mice during CsA treatment. Numbers of surviving CypA/wt (open circle) or CypA/ R55A (closed circle) transgenic mice or littermates (triangle) during CsA treatment were plotted for 7 or 28 days after CsA administration for short- or long-term experiment, respectively (P < 0:05).
Fig. 6. Effect of CypA/wt or CypA/R55A overexpression on expression of antioxidant enzymes in mice. Samples for Western (A) and Northern blot analyses (B) were prepared from kidneys of short- and long-term CsA-treated littermate, CypA/wt or CypA/R55A transgenic mice. Typical figures were demonstrated from three independent experiments. b-Actin was used as a loading control for Western blot. GAPDH transcript was used for RNA loading control.
Discussion treatment. Again, expression levels of transgenes in CypA/wt or CypA/R55A transgenic mouse lines appeared to determine the expression and activity levels of catalase after CsA treatment as observed in CsA-induced nephropathy (data not shown).
In this report, we first demonstrated in vivo that overexpression of CypA/wt can eliminate CsA-induced nephrotoxicity via CypA PPIase activity. The conclusion was evidenced by our observations that transgenic
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Fig. 7. Effect of CypA/wt or CypA/R55A overexpression on antioxidant enzyme activities in mice. Activity assays of catalase (A), CuZn-SOD (B), Mn-SOD (C), and GPx (D) from kidney samples of transgenic mice after CsA administration were performed as described in Materials and methods. Data represent means SE of three independent experiments. *; **P < 0:05, compared with littermate without CsA administration for short- or long-term experiment, respectively.
mice overexpressing CypA/wt were more resistant to CsA-induced nephrotoxicity, but those overexpressing CypA/R55A, a dominant negative mutant type of CypA PPIase activity, were more susceptible than littermates. In addition, the expression level of the transgenes, both CypA/wt and CypA/R55A, affected severity of CsA-induced nephrotoxicity. One possible explanation is that CsA titrates out CypA PPIase activity by forming a CsA–CypA complex when magnitude of PPIase activity determines the protective capacity against CsA. We have established the dosage-dependent protective effect of CypA PPIase on CsA-induced cytotoxicity more thoroughly in CypA/wt or CypA/R55A transfectants of MDCK renal tubular epithelial cells by observing that the ectopic expression level of either gene was directly proportional to the relieving or exacerbating capacity over the CsA-induced cytotoxicity (data not shown). ROS generation has been attributed to CsA-induced toxicity including nephrotoxicity [2–4]. Our finding that CypA/wt and CypA/R55A transfectants in MDCK cells (data not shown) as well as in H9c2 cardiac myoblasts [27] are more resistant or susceptible to H2 O2 , respectively, implies that CypA PPIase may function as an antioxidant. The anti-ROS function of CypA PPIase
activity derives an idea that cellular defense machinery against ROS might be a direct or indirect target of cis– trans isomerization. Also available are reports that CsA changes expression levels of different antioxidant enzymes [31,32]. To map out the target molecules, we determined alterations in the expression level and activity of antioxidant enzymes in our experimental animals in the absence or presence of CsA. Upon CsA treatment, catalase, but no other antioxidant enzymes, showed a dual regulatory pattern although absence of CsA negated the regulatory changes among littermate, CypA/ wt, and CypA/R55A transgenic mice: CsA down-regulated catalase expression in littermates, further in CypA/ R55A transgenic mice, but up-regulated it in CypA/wt transgenic mice. These data clearly exclude the possibility that catalase is a direct target of CypA PPIase activity. Simple explanation would be that mild oxidative stress positively regulates catalase expression but death decision by severe oxidative stress negatively regulates it. This can be supported by a recent report that low oxidative stress activates catalase expression through its interaction with c-Abl and Arg, while high level of ROS that evokes apoptosis has an antagonistic effect on catalase expression [33]. Indeed, our study in
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MDCK cells also displayed the dual regulatory mode between ROS generation and catalase expression. CsA and H2 O2 increased catalase expression in MDCK cells within the concentrations that cells could sustain, but decreased it beyond those concentrations in a dose-dependent manner (data not shown). Therefore, we surmise that catalase expression is regulated secondarily by intracellular ROS level rather than directly by CypA PPIase activity. At the present time, how CypA PPIase activity scavenges ROS remains to be elucidated. Inducibility of endogenous CypA has been reported under mild oxidative stress [34]. We also showed in the previous report that low CsA concentration induced endogenous CypA expression through generation of mild oxidative stress and the induced CypA provided resistance to higher CsA concentrations in H9c2 cardiac myoblasts [27]. MDCK cells showed the same effect of the CsA-CypA induction and resistance as H9c2 cells (data not shown). Interestingly, CypA induction has been reported in papillomavirus-immortalized oral keratinocytes and non-small cell lung cancer [35,36]. In addition, there are accumulating evidences that CypA expression is induced in diverse cells and tissues under different conditions, though the role of increased CypA expression has not been explored. CypA expression increases in mesenchymal stem cells [37], ultraviolet-radiated HeLa cells [38], and thrombinactivated platelets [39]. Importantly, our data show that overexpression of CypA is undisruptive as well as nontoxic to organ integrity including kidney in transgenic mice, excluding the possibility that overexpression of CypA is oncogenic or toxic to cell survival. Rather, it seems that CypA overexpression provides cells with more resistance to hypoxic or stress condition. As prevention of CsA toxicity is highly required in the clinics, our data may provide grounds for searching agents that induce CypA expression to relieve CsA nephrotoxicity. In conclusion, we suggest that CsA inhibition of CypA PPIase activity is a possible mechanism of CsAinduced nephrotoxicity. Besides, we showed that CypA overexpression is not only harmless in experimental animals but also protective against CsA-induced nephrotoxicity. To understand the molecular mechanism(s) of CypA PPIase as an antioxidant, we are currently trying to identify substrates of CypA PPIase. Acknowledgments We are grateful to Dr. Eric N. Olson at University of Texas Southwestern Medical Center for the critical reading of the manuscript and helpful discussion. We also thank Dr. Youn-Wha Kim at Kyung Hee University for technical assistance in histological analysis. This work was funded by grants from the Korea Science and Engineering Foundation (No. R13-2002-020-01001-0) and the Ministry of Public Health and Welfare (No. 03-PJ10-PG6-E001-0001), Republic of Korea.
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