http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, 2014; 24(6): 369–376 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2014.920447

RESEARCH ARTICLE

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Role of protein kinase A signaling pathway in cyclosporine nephrotoxicity F. D. Franc¸a1, A. F. Ferreira1, R. C. Lara1, J. V. Rossoni Jr2, D. C. Costa2, K. C. M. Moraes3, D. A. Gomes1, C. A. Tagliati4, and M. M. Chaves1 1

Departamento Departamento 3 Departamento 4 Departamento 2

de de de de

Bioquı´mica e Imunologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte – MG, Brasil, Cieˆncias Biolo´gicas, Instituto de Cieˆncias Exatas e Biolo´gicas, Universidade Federal de Ouro Preto, Ouro Preto – MG, Brasil, Biologia, Instituto de Biocieˆncias, Universidade Estadual Paulista ‘‘Ju´lio de Mesquita Filho’’, Rio Claro – SP, Brasil, and Ana´lises Clı´nicas e Toxicolo´gicas, Faculdade de Farma´cia Universidade Federal de Minas Gerais, Belo Horizonte – MG, Brasil

Abstract

Keywords

Cyclosporine is an important immunosuppressive agent; however, nephrotoxicity is one of the main adverse effects. The purpose of this study was to evaluate the effect of inhibiting the protein kinase A (PKA) signaling pathway in nephrotoxicity caused by cyclosporine from the assessment of cell viability, pro-inflammatory cytokines, and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Cyclosporine proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity assay (MTT), caused DNA fragmentation, determined by flow cytometry using the propidium iodide dye, and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cell lines, the production of IL-6 proved to be a dependent PKA pathway, while TNF-a was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by cyclosporine, and this production was dependent on the PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study’s results, it can be concluded that the inhibition of PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by cyclosporine.

Cyclosporine, IL-6, nephrotoxicity, NO, PKA, TNF-a

Introduction Cyclosporine is an important and powerful immunosuppressive agent that is widely used to prevent allograft rejection in solid organ transplantation (Behr et al., 2009; Hogan & Storb, 2004; Schrem et al., 2004) or to treat various autoimmune diseases (Abdel-latif et al., 2013), especially certain intractable nephrotic syndromes (Austin et al., 2009; Meyrier, 2009). Nonetheless, treatment with cyclosporine is limited due to its nephrotoxicity. Cyclosporine can induce both reversible and irreversible damages to all kidney compartments, including the glomeruli, arterioles, and tubulointerstitium (Naesens et al., 2009). Therefore, improving the understanding of the action mechanisms nephrotoxicity of cyclosporine is necessary.

Address for correspondence: Mı´riam Martins Chaves, PhD, Departamento de Bioquı´mica e Imunologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais – Caixa Postal 486, 30161-970 Belo Horizonte, Minas Gerais, Brasil. Tel: +55 31 3409 2660. Fax: +55 31 3409 2614. E-mail: [email protected]

History Received 2 October 2013 Revised 8 April 2014 Accepted 27 April 2014 Published online 4 August 2014

Renal cell lines have been employed as alternative methods for the study of therapeutic products that cause nephrotoxicity (Jung et al., 2009; Lincopan et al., 2005; Pfaller & Gstraunthaler, 1998; Price et al., 2004) and the use of in vitro techniques has enhanced the comprehension of molecular mechanisms of nephrotoxicity (Wilmes et al., 2011). The LLC-PK1 (porcine proximal tubular cells) and MDCK cells (canine distal cells) are considered acceptable models to study drug nephrotoxicity (El Mouedden et al., 2000; Ramseyer & Garvin, 2013; Servais et al., 2006; Shin et al., 2010; Yano et al., 2009; Yuan et al., 2011). The protein kinase A (PKA) signaling pathway is involved in the regulation of the cell functions in nearly all types of mammalian tissues, including the regulation of cell cycles, apoptosis, proliferation, and differentiation (Bichet, 2006). PKA kinase is a serine/threonine in its inactive form that consists of a tetramer composed of two regulatory subunits (R) and two catalytic subunits (C). Each R subunit contains two binding sites for the 3,5-cyclic adenosine monophosphate (cAMP), a second cellular messenger. Upon binding of the cAMP to the regulatory site, the dissociation of regulatory and catalytic subunits occurs, and two catalytic subunits are

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released, allowing them to catalyze the phosphorylation of proteins in regulatory residues (Gerits et al., 2008). In addition, signaling pathway involves the regulation of cytokine gene expression with the cAMP/PKA (GrandjeanLaquerriere et al., 2003). This pathway is also involved in the control of inflammatory process and vascular protection and its activation can affect a wide range of cellular events through the phosphorylation of cytoplasmatic and nuclear proteins (Kopperud et al., 2003). Cyclosporine impairs kidney function through multiple mechanisms including hypoxia, generation of oxidative stress and inflammation (Chander et al., 2005; Sa´nchez-Pozos et al., 2010), and altered expression of various inflammatory cytokines (Walker & Endre, 2008). Among the proinflammatory cytokines that were associated with the pathophysiology of nephrotoxicity highlight interleukin-6 (IL-6) and tumor necrosis factor (TNF-a) (Vianna et al., 2011). Furthermore, the renal vasoconstriction is attributed to an imbalance in the release of vasoactive substances including reduction factors in particular vasodilators as nitric oxide (NO) (Shihab et al., 2000; Yoon & Yang, 2009). NO has direct effects on vascular, inflammatory, and cellular processes (Hegarty et al., 2002), and plays an important role in the control of vascular tone and renal hemodynamics (Romero et al., 1992). Accordingly, many studies have been focused on the role of NO in cyclosporine nephrotoxicity (Abdel-latif et al., 2013), but this has not been well-defined. Many researchers have reported that cyclosporine impairs NO production (Chander & Chopra, 2005; Chander et al., 2005) whereas other studies have shown that NO synthesis is preserved or increased during cyclosporine nephrotoxicity (Bobadilla et al., 1998; Stroes et al., 1997). Therefore, it is controversial if the cyclosporine stimulates or not the production of NO. Thus, in the present work, the question has been raised as to whether or not nephrotocixity generated by cyclosporine depends on the PKA pathway signaling by evaluating cellular viability, pro-inflammatory cytokines, and NO production parameters.

Materials and methods Drugs Cyclosporine was kindly donated by Crista´lia (Produtos Quı´micos Farmaceˆuticos Ltda, Itapira, SP, Brazil). A stock solution of 500 mM of cyclosporine was prepared in a phosphate buffer saline (PBS) solution and different volumes were added to the RPMI-1640 medium (Sigma, St. Louis, MO) to generate eight different concentrations: 5, 10, 20, 25, 30, 40, 45, and 50 mM. The choice of cyclosporine concentrations was based on the findings from Nascimento et al. (2005). The inhibitor of PKA (H89) pathway (Calbiochem Merck KGaA, Darmstadt, Germany) was dissolved in anhydrous dimethylsulfoxide (DMSO) to form a concentrated solution that was 1000 times the required final concentration. The inhibitors were aliquoted and stored at 20  C. The concentrated solution was diluted immediately prior to use and the cells were pretreated with 1.0 mM of H89 for 30 min. The cellular viability with H89 was 93% (Chaves et al., 2009).

Toxicol Mech Methods, 2014; 24(6): 369–376

Cell culture The LLC-PK1 cell lines (kidney proximal tubular cells from pigs – passages 5–15), and MDCK (distal tubular cells from dogs – passages 5–15) were obtained from the Cell Bank at Universidade Federal do Rio de Janeiro (UFRJ). These were cultivated in an RPMI-1640 culture medium (Sigma, St. Louis, MO) and supplemented with 10% (v/v) bovine fetal serum (Invitrogen Co Ltd, Carlsbad, CA), 100 IU penicillin/ mL, and 100 mg streptomycin/mL (Sigma, St. Louis, MO). Cells were cultivated in 75 cm2 bottles and incubated at 37  C in a humidified with 5% CO2. Cell viability analysis Cell viability was determined by a quantitative colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Mosmann, 1983). The cells were placed on 96-well plates in a concentration of 5.0  103 cells/well. After 24 h incubation, 20 mL cyclosporine diluted with medium at full range concentrations were added to the wells for 1, 6, and 24 h. Having completed this exposure time, 20 mL of MTT solution (5.0 mg/mL) was added, and the plates were incubated for 1 h at 37  C. The MTT solution was then removed, and 100 mL of DMSO was added to each well. The absorbance was read at 570 nm (Thermo Plate model TP-READER, Thermo Scientific, Waltham, MA). Sub-diploid DNA content determination A flow cytometric DNA fragmentation assay was employed as a quantitative measure of cell death (Nicoletti et al., 1991). Twenty-four hours after treatment with cyclosporine, the cells were collected by centrifugation, lysed with 300 mL of a hypotonic solution containing 0.5% Triton X-100 and 50 mg/ mL propidium iodide (PI, Invitrogen, Carlsbad, CA). Cells were incubated at 4  C for 1 h and analyzed in a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany) for shifts in PI fluorescence that were indicative of nuclei with hypodiploid DNA content. To study the involvement in the inhibition of PKA pathway in cyclosporine induced cell death, LLC-PK1 and MDCK cells were pretreated for 30 min with 1.0 mM (H89-PKA inhibitor) followed by cyclosporine (5.0 mM) treatment. Subdiploid DNA content and cell viability were measured after 24 h to assess the cellular responses in the presence of the signaling pathway inhibitor. Western blot assay Cell culture bottles of MDCK and LLC-PK1 were grown on 75 cm2 bottles, with desired confluence and next cells were exposed to different substances in accordance with the following groups: negative control (untreated cells); positive control – 10 mM forskolin (Burgos et al., 2004) (Sigma-Aldrich, St. Louis, MO – SigmaÕ ); cells treated with cyclosporine (5.0 mM); cells treated with H89 (1.0 mM); and cells pre-treated with H89 for 30 min following cyclosporine (5.0 mM). After chemical additions, cells were incubated for 3 h at 37  C and 5% CO2. Next, cells were collected using the same methodology described above. After centrifugation, the supernatant was discarded and the precipitate was resuspended in

PKA and cyclosporine nephrotoxicity

500 mL of lysis buffer (Tris HCl 1.0 mM; EDTA 0.5 M; NaCl 5.0 M; DTT; Nonidet P40; protease inhibitor cocktail – SigmaAldrich, St. Louis, MO – SigmaÕ ). The homogenate was incubated on ice for 10 min, and next the samples were centrifuged for 2 h at 20 000 rpm at 4  C. The supernatant was collected and transferred to new microtube, and the proteins were finally quantified using Bradford assay in a spectrophotometer (spectrophotometer cuvette digital – Biosystems, Foster City, CA) at 595 nm (Bradford et al., 1976). Next, proteins were loaded in a 10% polyacrilamyde gel. After the electrophoresis, the proteins were electrotransfered onto a nitrocellulose membrane adapting the methodology described in Sambrook et al. (1989), and then incubated with the primary antibody (rabbit anti-PRKACG antibody polyclonal (Sigma-Aldrich, St. Louis, MO), with a dilution factor of 1:250 for 12 h at 4  C. After incubation, the membranes were extensively washed with TBST buffer (500 mmol/L NaCl, 20 mmol/L Tris-HCL, and 0.4% Tween 20; pH 7.4), followed by the secondary antibody incubation (anti-rabbit IgG-peroxidase, antibody produced in goat (Sigma-Aldrich, St. Louis, MO) for 2 h at 4  C. The secondary antibody was diluted by a factor of 1:10 000, and the same extensive washes with TBST were performed. Finally the bands were visualized after incubation with Luminol Enhancer Solution (GE Healthcare, Pittsburgh, PA) for 1 min, followed by a 15 s exposure to Hyperfilm-ECL (GE Healthcare). The results were quantified using Quantity One, BioRad, Hercules, CA.

LLC-PK1 and MDCK cells were incubated for 24 h into 24-well plates (5.0  105 cells/well) and, after this, they were pre-treated for 30 min with H89 (PKA inhibitor – 1.0 mM). After, the cells were treated with cyclosporine (5.0 mM) with and without H89 and the plates incubated again. After 24 h, supernatants were obtained by centrifugation 1500 rpm, 10 min, and NO production was measured by means the Griess reaction. This involved comparing 100 mL aliquots of culture supernatant with serial dilutions NaNO2 (from 7.81 mM to 1000 mM). To this, an equal volume of the Griess reagent (N-1-naphthylethylenediamine 0.1% in H2O + sulfanilamide 1% in 2.5% H3PO4) was added and then incubated at room temperature for 10 min and read at 540 nm (Green et al., 1982). Statistical analysis All results were analyzed by the one-way ANOVA and Tukey post-test using GraphPad Prism version 5.00 for windows (GraphPad Inc., San Diego, CA). p Value 50.05 was considered to indicate statistical significance.

Results Cytotoxic effects of cyclosporine in renal cell lines Cyclosporine proved to be cytotoxic for LLC-PK1 and MDCK cells (Figure 1). This toxicity could be observed after 1 h of exposure at concentrations of  10.0 mM in LLC-PK1 cell lines. However, in MDCK cells, only concentrations of 45 and 50 mM provoked a statistically significant difference after 1 h of exposure. After 6 and 24 h of exposure to cyclosporine, all concentrations (5.0–50 mM) caused significant reductions in cell viability; however, the cytotoxicity after 24 h was higher in both cell lines.

IL-6 and TNF-a levels in cell culture supernatants were performed in triplicate using commercially available highsensitivity enzyme-linked immunosorbent assay kit (Enzo Life Sciences, Inc, Plymouth Meeting, PA) according to the manufacturer’s instructions. LLC-PK1 and MDCK cells were plated at 5.0  105 cells/well into 24-well Plates. Twenty-four hours after cells were pretreated for 30 min with 1.0 mM (H89-PKA inhibitor), followed by cyclosporine (5.0 mM) treatment. After 24 h, supernatants cells were obtained by centrifugation 1500 rpm, 10 min, and were stored at 80  C. The sensitivities of each ELISA kit were 6.01 and 8.43 pg/mL for IL-6 and TNF-a, respectively.

DNA fragmentation induced by cyclosporine An increase in the percentage of dead cells occurred 24 h after treatment with cyclosporine. This cell population consists of a sub-diploid DNA content that is indicative of DNA 120

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DOI: 10.3109/15376516.2014.920447

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Toxicol Mech Methods, 2014; 24(6): 369–376

Western blot assay The results in Figure 4 show the involvement of the PKA signaling pathway in the nephrotoxicity of cyclosporine. Positive control (forskolin) and cyclosporine induced significant activation of PKA pathway in LLC-PK1 and MDCK cell lines (Figure 4, panels A and B). When cells were pretreated with H89 and then exposed to cyclosporine, a significant decrease in band intensity was observed compared with the group treated with cyclosporine (Figure 4, panels A and B). 25 Percentage of DNA fragmentaon

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Effects of protein kinase A inhibitor on production of IL-6 and TNF-a in renal cell lines The results showed that inhibition of PKA pathway (H89) significantly decreased in IL-6 production in LLC-PK1 cells (42%) and MDCK (37%). When cells were incubated with cyclosporine, an increase of 37% in IL-6 production and of 60% in LLC-PK1 and MDCK cells, respectively, could be observed. However, when the cells were treated with H89 and cyclosporine, a decrease in IL-6 production in LLC-PK1 cells (25%) and MDCK (50%) could be observed (Figure 5, panel A). For the production of TNF-a, incubation with H89 did not affect the production in any of the evaluated cells. When cells were incubated with cyclosporine, an increase in TNF-a production (29%) and (30%) by LLC-PK1 and MDCK cells, respectively, was observed. However, when the cells were pretreated with H89 associated with cyclosporine, no significant difference occurs in relation to the group treated with cyclosporine (Figure 5, panel B). Effects of protein kinase A inhibitor on the production of NO in renal cell lines Results from the present study showed that for the production of NO, incubation with H89 did not affect the production in any of the evaluated cells (Figure 6). In both cell lines, cyclosporine provoked a significant decrease in NO production: in LLC-PK1 cells, the NO decreased by 26%, whereas in MDCK cells, it fell by 70%. However, both cell lines were able to increase the production of NO significantly when they were pretreated with H89 associated with cyclosporine. In LLC-PK1 cells, the NO increased by 85%, as compared with 90% in MDCK cells (Figure 6).

Discussion

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Figure 2. DNA fragmentation induced by cyclosporine. The cells were placed at the density of 1.0  104 cells/well in a 24-well plate and were treated with cyclosporine (5.0 mM) in triplicates. DNA fragmentation was analyzed after staining with PI. A flow cytometric assay was employed as a quantitative measure of cell death. Results are expressed as a percentage of events from a total of 5000 events. Results represent mean + SD of triplicates from three independent experiments. Asterisk ¼ significantly different from control (p50.05).

Figure 3. Effect of pre-treatment with H89 on DNA fragmentation induced by cyclosporine (CsA). LLC-PK1 (A) and MDCK (B). The cells were placed at the density of 1.0  104 cells/well in a 24-well plate and were treated with cyclosporine with or without H89 pre-treatment (1 mM, 30 min). DNA fragmentation was analyzed after staining with PI. A flow cytometric assay was employed as a quantitative measure of cell death. Results are expressed as a percentage of events from a total of 5000 events. Results represent mean + SD of triplicates from three independent experiments. * and # were significantly different from control and group treated with cyclosporine (p50.05).

The involvement of the PKA signaling pathway in the nephrotoxicity of cyclosporine has yet to be fully studied. To search for new strategies to prevent/reduce nephrotoxicitiy, in vitro assays were performed to evaluate the participation of the PKA pathway. As shown in Figure 1, the renal toxicity of cyclosporine is time and concentration dependent and proved to be greater after 24 h of exposure. Furthermore, the present study’s results demonstrated that cells from different regions of the nephron (LLC-PK1 and

(A) Percentage of DNA fragmentaon

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fragmentation and cell death. This alteration could be observed in LLC-PK1 and MDCK cell lines. The quantitative analysis of DNA fragmentation in two lineages is demonstrated in Figure 2. LLC-PK1 cells presented 7.94% of cell death, while the MDCK cells presented 14.19%. Pretreatment with H89 failed to reduce the fragmentation of DNA provoked by cyclosporine in LLC-PK1 cells (Figure 3, panel A). In MDCK cells, the inhibition of the PKA pathway caused a significant reduction in DNA fragmentation (Figure 3, panel B), which was assayed by determining the sub-diploid DNA content after 24 h of treatment with cyclosporine (Figure 3).

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Figure 4. PKA activation by cyclosporine in cells lines LLC-PK1 and MDCK – Western blot assay. Positive control: forskolin (10 mM); CsA ¼ cyclosporine; H89 + CsA ¼ H89 + cyclosporine. *p50.05 for values significantly different from the negative control groups. #p50.05 for values significantly different from the groups treated with cyclosporine. The statistical was first analyzed by image analysis software Quantity One, BioRad, Hercules, CA, and posteriori by the ANOVA and Tukey post-test.

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Figure 6. Effects of cyclosporine on nitric oxide (NO) production in renal cell lines. The production of NO in the LLC-PK1 and MDCK supernatant cultures was determined by the Griess reaction after 24 h. The cells were treated with cyclosporine with or without H89 pre-treatment (1 mM, 30 min). The results represent the mean ± SD of the results of three independent experiments performed in sextuplicate. *p50.05 when compared with the negative control group (untreated cells). #p50.05 relative to the cyclosporine group.

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Figure 5. Effects of cyclosporine on IL-6 and TNF-a production in renal cell lines. The production of each cytokine, IL-6 (A) or TNF- a (B), in the LLC-PK1 and MDCK supernatant cultures was determined by ELISA after 24 h. The cells were treated with cyclosporine with or without H89 pre-treatment (1 mM, 30 min). The results presented in A and B represent the mean ± SD of the results of three independent experiments performed in duplicate. *p50.05 when compared with the negative control group (untreated cells). #p50.05 relative to the cyclosporine group.

MDCK) present varying sensitivities to the cytotoxic effects of cyclosporine. According to Wilmes et al. (2011), due to the functional and biochemical heterogeneity of nephrons, susceptibility to toxicity can vary among nephron segments. The MTT assay is mainly based on the enzymatic conversion of MTT in the mitochondria (Fotakis & Timbrell, 2006) and many studies have demonstrated the considerable involvement of the mitochondria in the mechanisms of nephrotoxicity (Rodriguez-Enriquez et al., 2004; Servais et al., 2006). Therefore, one can infer that cyclosporine promotes changes in mitochondrial metabolism, considering that the MTT evaluates the integrity of this organelle. According to Peyrou et al. (2005), the imbalance of intracellular calcium homeostasis, protein alkylation, and cellular oxidative stress are described as initial changes observed in renal cells

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exposed to nephrotoxic compounds. Mitochondria, endoplasmatic reticulum, lysosomes, and cell membranes are identified as targets. Therapeutic concentrations of cyclosporine range from 0.1 to 1.6 mM (Hauser et al., 1998; Kovarik et al., 2003). Jiang & Acosta (1993) also reported that due to its high lipophilicity, cyclosporine mainly accumulates in the kidneys, with concentration levels reaching as much as 50 times that found in blood plasma. Therefore, the present study opted for the use of a 5.0 mM concentration of cyclosporine in studies that evaluate the involvement of the PKA pathway in nephrotoxicity. The increased fragmentation of DNA observed in flow cytometry (Figure 2) can be interpreted as cell death (Nicoletti et al., 1991). Therefore, it can be concluded that cyclosporine caused cell death in the two studied cell lines, and these can be found in the late stages of apoptosis/necrosis. Propidium iodide (PI) is widely used in the study of cell death, as it does not penetrate through the cell membrane, thus differentiating among normal cells of apoptotic and necrotic cells. A characteristic of the cells in the early stages of apoptosis is the maintenance of the integrity of the membrane and the ability to exclude dyes, such as PI (Aubry et al., 1999). Late phases of apoptosis are commonly accompanied by an increased permeability of the cell membrane, which allows for an intake of PI within the cells (Hashimoto et al., 2003). Apoptosis has been clearly shown in tubular and interstitial cells of transplant patients with chronic cyclosporine nephrotoxicity (Hauser et al., 2005). Tubular cell apoptosis can also be observed in animal and cell culture models (Amore et al., 2000). Cyclosporine-induced apoptosis is primarily triggered through the mitochondrial pathway. The generation of reactive oxygen species (indirectly demonstrated in vitro in tubular epithelial cells through the protective effect of prednisone; Jeon et al., 2005), the reduction of the Bcl-2 expression, the increased expression of Bax (in mesangial cells as well as in vivo) (Han et al., 2006), and the translocation of Bax to the mitochondria (Justo et al., 2003) all contribute to the induction of apoptosis. Moreover, in cultured tubular cells, caspases 2, 9, and 3 are directly activated by cyclosporine (Tarze et al., 2007). After having evaluated the cell death caused by cyclosporine in LLC-PK1 and MDCK cells, the present study aimed to assess whether or not the inhibition of PKA pathway could influence cell death detected in this study, given that this pathway is directly linked to cell cycles and survival. In this context, this study’s results showed that in the LLC-PK1 cells’ inhibition of PKA pathway did not alter the DNA fragmentation caused by cyclosporine (Figure 3, panel A). In the MDCK cell, the inhibition of PKA pathway reduced cell death caused by cyclosporine (Figure 3, panel B). In B cells, the activation of PKA caused a reduced expression of the anti-apoptotic protein Mcl-1, which is associated with apoptosis (Myklebust et al., 1999). In contrast, PKA activation can result in the phosphorylation of Bad – at the same site as that induced by Raf and MEK – and is associated with the anti-apoptotic effects of PKA (Harada et al., 1999). Effects of PKA on apoptosis are likely

Toxicol Mech Methods, 2014; 24(6): 369–376

to be largely dependent on the cell type and the mechanisms by which apoptosis is induced (Franklin & McCubrey, 2000). To confirm that cyclosporine induces the activation PKA pathway, the Western blot assay was used (Figure 4, panels A and B). In both cell lines, the group of cells treated with forskolin (adenylyl cyclase stimulator) and the group treated with cyclosporine presented a significant difference in relation to the negative control (untreated cells). Another indication of the activation of the PKA pathway was found when a significant difference could be observed when comparing the group treated with cyclosporine and H89 with the group treated only with cyclosporine. H89 is highly selective inhibitor of the PKA pathway (Bracken et al., 2006). Therefore, the study’s results suggest that nephrotoxicity of cyclosporine may well be related to decreased cell viability, DNA fragmentation, and that the inhibition of PKA signaling pathway can aid in decreasing this toxic effect. Other studies have shown that the use of a PKA inhibitor eliminated lipid peroxidation and cellular injury induced by cephaloridine (Kohda & Gemba, 2001). In addition, inflammation plays a key role in the pathogenesis of drug-induced kidney injury (Chai et al., 2013). In both cell lines, the production of IL-6 proved to be dependent on the PKA pathway (Figure 5, panel A), and TNF-a was not influenced by the inhibition of the PKA pathway (Figure 5, panel B). These results also suggest the hypothesis that the inhibition of the PKA pathway can aid in reducing the nephrotoxicity of cyclosporine, given that the proinflammatory cytokine IL-6 proved to be associated with the pathophysiology of nephrotoxicity (Vianna et al., 2011). Specifically in renal tissue, cytokines induce local proliferation of tubular and interstitial cells, extracellular matrix synthesis, procoagulant activity of the endothelium, formation of reactive oxygen species, and increased expression of adhesion molecules and biologically active lipids (Rao et al., 2007). The PKA pathway phosphorylates the NF-kB p65 subunit leading to NF-kB activation. Binding of the activated NF-kB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes (Wang et al., 2011). Therefore, it can be inferred that decreased production of IL-6 in renal cell lines with the PKA pathway may be inhibited due to the absence of NF-kB activation. When evaluating the production of TNF-a, cyclosporine significantly increased its production in both cell lines (Figure 5, panel B). However, this production was not altered when the PKA pathway was inhibited. Many researchers have reported that renal ischemia leads to an increase in renal TNF-a levels (Donnahoo et al., 2000; Gabr et al., 2011) commonly associated with cyclosporine administration (Abdel-latif et al., 2013). Furthermore, the oxidative stress and the products resulting from the lipid peroxidation most likely serve as activators of transcription factors, in turn leading to the induction of the gene expression of proinflammatory cytokines and the release of many inflammatory cytokines, including TNF-a (Mariappan et al., 2007). In this context, in both studied cell lines, NO production was increased when cells were pre-incubated with H89, followed by cyclosporine, and this production proved to be

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DOI: 10.3109/15376516.2014.920447

dependent on the PKA pathway (Figure 6). NO is formed from the amino acid L-arginine by means of NO synthases (NOSs). Efforts to supplement NO levels with L-arginine have been shown to afford protection to the obstructed kidney, in which vasodilatory actions of NO are likely to be involved (Hegarty et al., 2002). It is possible that the deficiency in NO below normal physiological level may activate other proinflammatory mediators (Colasanti & Suzuki, 2000). Previous studies have proposed renal tubular injury as a possible consequence of renal vasoconstriction and endothelial injury leading to ischemia, as well as a direct toxic effect of cyclosporine on tubular epithelium (Zhu et al., 2012). The PKA pathway is cited in the literature as a participant in the phosphorylation of NOS (Genestra et al., 2001) and, depending on the NOS, isoform of the phosphorylated form is inactive. Komeima et al. (2000) showed that the phosphorylation of NOS leads to a decrease in NOS activity. Martinez-Mier et al. (2000) reported that NO demonstrated a protective effect in ischemic kidney injury and exerted an antiapoptotic action by downregulating the expression of p53 (Du et al., 2007). NO-mediated up-regulation of Bcl-2/Bax ratio and attenuation of cytochrome c release (Kukreja et al., 2005) and cyclosporine-induced apoptosis are partially mediated by NO inhibition (Xiao et al., 2012). Therefore, it can be concluded that the inhibition of PKA may have a beneficial effect in preventing nephrotoxicity of cyclosporine through the vasodilator effect of NO.

Conclusions These findings can serve to produce alternative in vitro methodologies that can help to discover therapeutic procedures as well as to create a more well-rounded understanding of the molecular mechanisms involved in the processes of nephrotoxicity. Thereby, considering the present study’s results as a whole, it can be concluded that therapies that use the inhibition of PKA signaling pathways may help in reducing the degree of nephrotoxicity caused by cyclosporine.

Declaration of interest Pro´-Reitoria de Pesquisa da Universidade Federal de Minas Gerais, UFOP (Universidade Federal de Ouro Preto) FAPEMIG (Fundac¸a˜o de Amparo e Pesquisa de Minas Gerais – Process APQ 00596-08), CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior) and CNPq (Conselho Nacional de Pesquisa) supported this paper.

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