Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats

Tan Yong Chia; Munavvar Abdul Sattar; Ahmad F Ahmeda; Nurzalina Abdul Karim Khan; Vikneswaran Murugaiyah; Ashfaq Ahmad; Zurina Hassan; Gurjeet Kaur; Mohammed Hadi Abdulla; Edward James Johns

doi:10.1371/journal.pone.0231472

. 2020 Apr 16;15(4):e0231472. doi: 10.1371/journal.pone.0231472

Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats

Tan Yong Chia ^1,^*, Munavvar Abdul Sattar ¹, Ahmad F Ahmeda ², Nurzalina Abdul Karim Khan ¹, Vikneswaran Murugaiyah ¹, Ashfaq Ahmad ^3,⁴, Zurina Hassan ⁵, Gurjeet Kaur ⁶, Mohammed Hadi Abdulla ⁷, Edward James Johns ⁷

Editor: Partha Mukhopadhyay⁸

PMCID: PMC7161975 PMID: 32298299

Abstract

Oxidative stress is involved in the pathogenesis of a number of diseases including hypertension and renal failure. There is enhanced expression of nicotinamide adenine dinucleotide (NADPH oxidase) and therefore production of hydrogen peroxide (H₂O₂) during renal disease progression. This study investigated the effect of apocynin, an NADPH oxidase inhibitor and catalase, an H₂O₂ scavenger on Cyclosporine A (CsA) nephrotoxicity in Wistar-Kyoto rats. Rats received CsA (25mg/kg/day via gavage) and were assigned to vehicle, apocynin (2.5mmol/L p.o.), catalase (10,000U/kg/day i.p.) or apocynin plus catalase for 14 days. Renal functional and hemodynamic parameters were measured every week, and kidneys were harvested at the end of the study for histological and NADPH oxidase 4 (NOX4) assessment. Oxidative stress markers and blood urea nitrogen (BUN) were measured. CsA rats had higher plasma malondialdehyde (by 340%) and BUN (by 125%), but lower superoxide dismutase and total antioxidant capacity (by 40%, all P<0.05) compared to control. CsA increased blood pressure (by 46mmHg) and decreased creatinine clearance (by 49%, all P<0.05). Treatment of CsA rats with apocynin, catalase, and their combination decreased blood pressure to near control values (all P<0.05). NOX4 mRNA activity was higher in the renal tissue of CsA rats by approximately 63% (P<0.05) compared to controls but was reduced in apocynin (by 64%), catalase (by 33%) and combined treatment with apocynin and catalase (by 84%) compared to untreated CsA rats. Treatment of CsA rats with apocynin, catalase, and their combination prevented hypertension and restored renal functional parameters and tissue Nox4 expression in this model. NADPH inhibition and H₂O₂ scavenging is an important therapeutic strategy during CsA nephrotoxicity and hypertension.

Introduction

Cyclosporine A (CsA) is a potent immunosuppressant agent that is widely used in solid organ transplantation. However, CsA use is associated with several unwanted side effect including nephrotoxicity and hypertension [1]. The incidence of chronic kidney disease (CKD) due to the use of calcineurin inhibitors (CNIs) such as CsA was reported in a cohort study of non-kidney transplant recipients (mostly liver, heart, and lung) [2]. In the mentioned study, CsA was given to 60% of the patients and was followed for 36 months. At the end of the study, 17% of the participants developed CKD (defined as an estimated GFR ≤29 mL/min/1.73 m²). The exact mechanism for CsA induced nephrotoxicity remains obscure. However, its immunosuppressive action involves intracellular interaction with calcineurin phosphatase that reduces the production of interleukin-2 (IL-2) which is an inflammatory cytokine. Initially, CsA binds to a specific family of cytoplasmic receptors known as cyclophilins and forms a complex that binds to and inhibits calcineurin, a transcription factor within T-lymphocytes cytoplasm [3, 4]. CsA prevents the de-phosphorylation of calcineurin and blocks the cellular mechanisms required for up-regulation of mRNA expression. These effects of CsA regulate the synthesis of many lymphokine mediators such as IL-2 and interferon-ɤ which is critical for T-lymphocyte proliferation and maturation, and macrophage activation [5].

CsA induced nephrotoxicity has been extensively reported in humans [6–8]. Patients treated chronically with CsA showed decreased glomerular filtration and renal perfusion accompanied by reduced proximal tubules reabsorptive capacity [6]. In another study, there was an increase in renal vascular resistance due to elevated levels of thromboxane in renal allograft recipients treated with CsA [7]. On another hand, CsA induced nephrotoxicity in experimental animals was extensively studied. Literature reported on CsA induced nephrotoxicity demonstrated that renal function can be compromised and a reduction of glomerular filtration and creatinine clearance was reported before in rats [9]. Moreover, CsA mediated water and sodium retention was shown to contribute to the development of hypertension in this model of renal injury in dogs [10]. Activation of renin-angiotensin-aldosterone via the effects of angiotensin II on angiotensin Type 1 (AT₁) receptors produced renal vasoconstriction and promoted renal tissue fibrosis in rats [5, 11]. The renal vasoconstriction can cause to tissue hypoxia and enhance the formation of reactive oxygen species that ultimately cause cellular injury and apoptosis.

NADPH oxidase (NOX) enzyme catalyzes the transfer of electrons from NADPH to molecular oxygen and generates oxygen free radical (O₂^-). It was suggested that NOX4 is the predominant source of O₂^- generation in the renal cortex homogenate [12]. Under physiological conditions, O₂^- present at low levels due to the scavenging activity of superoxide dismutase (SOD) enzyme which converts O₂^- into H₂O₂. The later itself is a bioactive molecule that can be broken down to H₂O by intracellular catalase enzyme. Although NOX4 have low levels of constitutive activity normally but in response to cytokines [13] and growth factors [14], NOX activity can increase significantly. Thus, NOX-generated O₂^- becomes exceedingly greater than endogenous antioxidant defence capacity and this leads to oxidative stress and tissue injury. As reviewed before, NADPH oxidase homologues are differentially expressed in endothelial, smooth muscle and infiltrating immune cells [15] indicating a wide range of effects at different levels in these cells.

Human studies reported the effects of NOX4 on vascular tissues such as coronary and peripheral arteries with contribution to atherosclerosis and endothelial dysfunction in these vessels [16, 17]. However, the pattern of and physiological importance of NOX4 expression in human kidney is not fully elucidated. A clinical study depicted the presence of NOX5 in the renal vasculature [18], while another study showed correlation between NOX5 expression in the kidney and tissue injury in diabetic nephropathy [19]. The aetiology of excessive NOX4 during disease was found to be related at least in part to higher activity of transforming growth factor-beta (TGF-β) in a variety of cell types including endothelial and kidney cells [20, 21]. Similarly, it was also reported that tumor necrosis factor-alpha (TNF-α) enhances the expression of NOX4 in a variety of vascular cells [22].

A previous study on rats revealed that apocynin significantly ameliorates inflammatory response and terminates the disease process by inhibiting NOX4. It was suggested that apocynin significantly down regulates NOX expression via activated PI3K/Akt and glycogen synthase kinase-3β (GSK-3β) pathway which is involved in the regulation of cellular inflammation and oxidative stress [23]. However, the role of NADPH oxidase in the mechanism of kidney injury and inflammation due to CsA is not fully understood. We hypothesized that in a model of CsA-induced renal injury and inflammation inhibition of NADPH oxidase by apocynin and H₂O₂ scavenging by catalase can ameliorate kidney injury and restore renal excretory function. Renal function and haemodynamics, and expression of NOX4 in kidney homogenate were examined in CsA rats following 14 days treatment with apocynin, catalase, and their combination.

Materials and methods

Animals

The study was performed in accordance with the guidelines of Animal Care and Use Committee in Universiti Sains Malaysia (USM) with the approval letter number USM/IACUC/2017/(106)844. Animal procedures were done under The Malaysian Code for the Care and Use of Animals for Scientific Purposes (MyCode) adapted from the Australian Code for the Care and Use of Animals for Scientific Purposes 8th Edition published by the National Health and Medical Research Council of Australia. Experiments were performed on a total of 64 adult male Wistar-Kyoto rats weighing 200-250g. Animals were procured from the Central Animal Facility of USM, Penang, Malaysia and were housed in the Animal Care Facility at the School of Pharmaceutical Sciences, USM, Penang, under a 12-h light-dark cycle, a temperature of 24°C and a humidity of 60%. Rats were provided with commercial rat 56chow (Gold Coin Sdn. Bhd., Penang, Malaysia) and tap water ad libitum. All animals were randomly assigned into two main groups namely, a CsA-induced renal failure group (25 mg/kg/day via gavage, Neoral^®, Novartis, Basel, Switzerland) and a control group (receiving the same volume of distilled water via gavage). Each of these groups was then sub-divided into four groups (n = 8) based on whether treated or not with apocynin (2.5 mmol/L p.o.), catalase (10000 U/Kg/day i.p.), and a combination of apocynin and catalase (Sigma-Aldrich, St. Louis, Missouri, United States) once daily for 14 days. Study was terminated on day 14 where acute experiments were performed on rats and kidneys were harvested for histopathology.

Metabolic and renal functional studies

Weekly metabolic data were collected throughout the study using metabolic cages (Nalgene^®, Thermo Scientific, Philadelphia, USA) where animals were kept for 24 hours. Body weight, water intake and urine output were recorded. Blood samples (500 μl) were obtained from the tail vein. Blood and urine samples were centrifuged at 3500 rpm for 10 minutes and the supernatants were collected before being stored at -30°C for later biochemical analysis. Plasma and urine creatinine (Jaffe’s reaction), and urinary protein levels (Bradford’s Assay) were measured spectrophotometrically (PowerWave X340, BioTek Instrument Inc., Vermont, USA) while plasma and urinary sodium and potassium concentrations were measured using a flame photometer (Jenway, PFP-7, England, UK). Creatinine clearance (CrCl), fractional sodium excretion (FE_Na⁺) and urinary protein excretion were calculated using standard equations as previously reported [9]. The BUN in this study was determined by urease/glutamate dehydrogenase method using a biochemical analyser (Biosystem^®, Shanghai, China). Methodology for measuring BUN was followed as directed by manufacturer.

Systolic blood pressure measurement

Weekly non-invasive blood pressure was measured using the CODA^® tail cuff plethysmography method (Kent Scientific Corporation, Torrington, CT, USA). Plethysmography utilises blood volume changes in the tail to detect blood pressure. Prior to entering the experimental protocol, the animals were subjected to three-day acclimatization. During each session, a total of 10 consecutive readings were selected from each rat and average values were determined.

Hemodynamic study

General preparation and surgical procedure

General surgical procedures were performed according to established protocols from this laboratory [24]. Overnight-fasted rats were anesthetized with an i.p. injection of 60 mg/kg sodium pentobarbitone (Nembutal^®, CEVA, Santé Animale, Libourne, France). Tracheotomy (PE240, Portex, Kent, UK) was performed through a small incision to facilitate respiration and the left jugular vein was cannulated (PE50, Portex, UK) to enable supplemental anaesthesia administration (15 mg/kg in 0.9%NaCl) as necessary. The right carotid artery was cannulated (PE50) and pushed up to the level of the aortic arch to allow continuous blood pressure recording using a fluid filled pressure transducer (P23 ID Gould, Statham Instrument, London, UK). A midline incision was made in the anterior abdomen to expose the left kidney and iliac artery. A cannula (PE50) was inserted into the iliac artery past the iliac bifurcation into the abdominal aorta to the level of renal artery such that its bevelled tip faced the entrance of the renal artery to enable direct delivery of saline (9g/L NaCl) at a rate of 6 ml/kg/h using a perfusion pump (Perfusor Secura FT 50mL, B. Braun Medical AG, Sempach, Switzerland) throughout the entire study period. A second pressure transducer was attached to the renal arterial cannula and both pressure transducers from the carotid and the renal arteries were attached to a computerized data acquisition system (PowerLab^®, AD Instruments, Sydney, Australia) for concomitant and continuous blood pressure recordings. A laser Doppler flow probe (OxyFlow, ADInstruments, Sydney, Australia) linked to a blood flowmeter was place on the left kidney capsule for renal cortical blood perfusion (RCBP) measurement.

Measurement of pulse wave velocity

Pulse wave velocity (PWV) was measured as previously reported [24]. PWV is a widely used marker of arterial stiffness and is studied in many pathologies such as left ventricular hypertrophy [25], hypertension [26, 27], renal failure [24] and diabetes [28]. In humans, pulse wave velocity is utilized as a predictor of cardiovascular events and mortality [29]. Upon completion of the surgical procedures, a stabilization period of one hour was given to allow equilibrium. Pulse pressure wave was recorded at a sampling rate of 400 Hz for 30 min. At the end of the experiment, animals were euthanized with an overdose (200 mg/kg) of sodium pentobarbitone and full contour of the aorta was exposed. The propagation distance (d) between the tips of the two cannulas was measured by locating a damp thread over the contour of the aorta. Whereas, the propagation time (t) for the blood pressure wave which moves from the aortic arch to abdominal aorta was measured manually using the time delay between the upstrokes (foot) of each pressure wave front by employing “foot to foot’ technique [30]. The values of PWV were calculated by dividing d by t and expressed in units of meters per second. β-index was calculated to evaluate the participation of diastolic blood pressure (DBP) in increasing arterial stiffness. It was calculated using the following formula (β-index = 2.2×(PWV)²/DBP) according to a previous report [31].

Biochemical analysis for oxidative stress marker

At the end of the acute experimental protocol, a 3 mL arterial blood sample was withdrawn via the carotid artery cannula and centrifuged at 3500 rpm for 10 minutes. The plasma samples were then stored at -30°C for further analysis of oxidative stress markers. Furthermore, kidney tissues were extracted at the end of the acute experiment and processed for oxidative stress biomarkers according to manufacturer’s guidelines. The extent of lipid peroxidation was accessed by measuring malondialdehyde (MDA) formation using the thiobarbituric acid reaction method [32]. Malondialdehyde reacts with thiobarbituric acid in acidic medium to give a pink-coloured pigment at 95°C. Superoxide dismutase (SOD) activity in the plasma was determined spectrophotometrically using an assay kit via a method established by Oyanagui [33]. SOD activities in the samples were determined by hydroxylamine assay developed from xanthine oxidase assay. Briefly, the test principle is as follows: superoxide anions are generated by xanthine and xanthine oxidase system. These superoxide anions oxidize hydroxylamine leading to formation of nitrite. This nitrite reacts with naphthalene diamine and sulfanilic acid to produce a coloured product. Indirect measurement of nitric oxide (NO) activity was done using a method described in a previous study [34] which involved a reaction between nitroxides and sulfanilic acid, and N-(1-naphthyl) ethylenediamine that generates a coloured product that can be detected using spectrophotometry. In the present study, the principle test was done according to nitrate reductase method. Since the final stable end product of NO in vivo are NO₂^- and NO₃^-. Thus, the total of both NO₂^- and NO₃^- was determined as an index of total NO production. The total NO concentration in the samples was done according to Griess method [35]. Lastly, total antioxidant capacity (T-AOC) was quantified by a method reported by Miller et al [36] where a reaction between 2,2’-azinobis-(3- ethyl-benzothiazoline-6-sulphonic acid) and peroxidase results in a relatively stable radical cation which upon interaction with Ferryl Myoglobin produces a relatively stable product that can be measured spectrophotometrically. The principle is based on the inhibition of 2, 2’-Azino-di-[3-ethylbenzthiazdine sulphonate] radical (ABTS^R) by antioxidants in the plasma. Radical cation ABTS^R+ was generated by incubation of ABTS^R with a peroxidase (metmyoglobin) and H₂O₂. All assays were carried out according to the manufacturer guidlines (NJJC Bio, Nanjing JianCheng, Bioengineering Institute, China).

Histopathological studies

The left kidney was carefully isolated from adipose and connective tissues. The excised kidney was then blotted dry on a laboratory filter paper and preserved in 10% neutral buffered formalin solution until histological examination. All tissues underwent a procedure reported using Haematoxylin and Eosin (H&E) staining [9, 27]. Histology was examined by a pathologist in this university (Dr G. K.). Kidney index (KI) was calculated using a standard equation (Kidney index = kidney weight / body weight x 100).

Relative quantification of NOX4 mRNA expression in the kidney using StepOnePlus RT-PCR system

The contralateral kidney was harvested and stored in RNAlater solution (Ambion, Life Technologies, Pleasanton, CA, USA) at -80°C in order to sustain the RNA integrity until further procedure. The quantitative RT-PCR reaction was performed on all eight experimental groups with a total of 64 rat kidney samples. Each rat kidney sample was further analysed in a triplicate manner. The extraction procedure was performed under a sterile environment. All equipment (harvesting desk, beaker, tissue, test tubes, surgical blades, and scissors) was cleaned with RNAZap^® solution (Ambion, Life Technologies Corporation, USA) to prevent any possible contamination. TRIzol reagent (Ambion, Life Technologies Corporation, USA) was used to extract RNA from kidney tissue according to the manufacturer’s guidelines. Upon various sequential steps of homogenization, washing and elution, total RNA was extracted, optimized, and quantified for purity using a NanoDrop^™ Lite UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) followed by total RNA to cDNA conversion using a high capacity RNA-to-cDNA kit (Applied Biosystems, Waltham, MA, USA). A volume of 20 μl of RNA was used for the conversion of cDNA using the default setting of the StepOnePlus RT-PCR system (Applied Biosystems, Singapore). Of the 20 μl, 11 μl comprised kit components (2× buffer, 10 μl; 20× enzymes, 1 μl), and the remaining 9 μl consisted of total RNA (depending upon the yield). TaqMan primers and probes for Nox4 gene (GenBank Accession N0. AY027527.1 and Rn00585380_m1) were derived from TaqMan-Gene Expression assays (Applied Biosystems, Waltham, MA, USA) [37]. Similarly, TaqMan primers and probes for β-actin gene (endogenous control, GenBank Accession N0. NM 031144.3 and Rn00667869_m1) were also derived from TaqMan-Gene Expression assays (Applied Biosystems, Waltham, MA, USA).

TaqMan Gene Expression assays were performed according to the manufacturer’s protocol. The amplification began with a total 20 μl reaction mixture. One RT-PCR reaction consisted of 10 μl of TaqMan Fast Master Mix (2×); 1 μl of TaqMan Gene Expression assays (20×) of the respective genes Nox 4 and β-actin; 8 μl of RNase-free water (Invitrogen, Carlsbad, CA, USA); and 1 μl of unknown sample cDNA. Temperature settings were done according to the manufacturer’s default settings. The amplification reactions were carried out on a MicroAmp^® Fast 96-well reaction plate (0.1 ml, Applied Biosystems, Life Technologies).

Quantitative RT-PCR reactions were performed on renal cortex of the extracted kidneys. Amplification of the housekeeping enzyme β-actin (endogenous control) allowed sample loading and normalization to be determined. The relative quantification of the target gene Nox4 and β-actin was calculated using the comparative C_T (threshold cycle) method with arithmetic formula (2^-ΔΔCT) [38].

The statistical analysis for the study was done using GraphPad Prism Version 5 software (GraphPad Software, San Diego, California, USA). All data retrieved from metabolic, renal functional, oxidative stress markers, haemodynamics and molecular studies were analysed with repeated measure one-way ANOVA followed by Bonferroni post hoc test. All data were presented as mean ± S.E.M with significance at P<0.05 level.

Results

Apocynin and catalase ameliorated reduction in body weight and water intake in CsA rats

The daily intake of food and water was similar across all experimental groups but there was a reduction (P<0.05) in body weight gain in the CsA group. Treatment of CsA rats with apocynin and catalase enhanced body weight gain compared to untreated rats by the end of the treatment period (Fig 1). There was a decrease in water intake (P<0.05) in CsA rats compared to control rats at day 7 and 14 of the study period. However, treatment of CsA rats with apocynin and apocynin plus catalase but not catalase alone resulted in greater water intake compared to untreated rats (Fig 2).

Fig 1 — CsA, Cyclosporine A. Data presented as mean±SEM. * p<0.05 from Day 0; # p<0.05 of Cx vs. C (Day 14); ¥ p<0.05 of all groups vs. C except Cx (Day 14); † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx (Day 14); § p<0.05 of CxApo vs. CxApoCat (Day 14); ¶ p<0.05 of CxCat vs. CxApoCat (Day 14); δ p<0.05 of CxCat vs. CxApo (Day 14).

Fig 2 — CsA, Cyclosporine A. Data presented as mean±SEM. * p<0.05 from Day 0; # p<0.05 of Cx vs. C (Day 14); ¥ p<0.05 of all groups vs. C except Cx (Day 14); † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx (Day 14).

Apocynin and catalase restored renal excretory function in CsA rats

Urine volume did not change during the study period in all groups except for CsA rats where a significant reduction (P<0.05) in urine excretion compared to baseline was observed on day 7 and remained low on day 14 of the study. Treatment of CsA rats with apocynin, catalase, and a combination of apocynin and catalase restored urine output to near control values (Fig 3).

Fig 3 — CsA, Cyclosporine A. Data presented as mean±SEM. * p<0.05 from Day 0; # p<0.05 of Cx vs. C (Day 14); † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx (Day 14).

CsA resulted in a decreased (P<0.05) creatinine clearance from baseline value (day 0) on day 7 and 14 of the study. The creatinine clearance in CsA group on day 14 of the study was smaller (P<0.05) compared to respective value in control group. The treatment of CsA rats with apocynin and a combination of apocynin and catalase increased creatinine clearance to values comparable to control at the end of treatment period as shown in Fig 4.

Fig 4 — CsA, Cyclosporine A. Data presented as mean±SEM. * p<0.05 from Day 0; # p<0.05 of Cx vs. C (Day 14); ¥ p<0.05 of all groups vs. C except Cx (Day 14); † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx (Day 14); § p<0.05 of CxApo vs. CxApoCat (Day 14); ¶ p<0.05 of CxCat vs. CxApoCat (Day 14); δ p<0.05 of CxCat vs. CxApo (Day 14).

The fractional excretion of sodium (Fig 5) and urinary sodium to potassium ratio (Fig 6) were not changed in control rats, those treated with apocynin, catalase, and their combination. However, in CsA treated rats, there was a significant reduction (P<0.05) in these parameters at day 7 and 14 of the study. Administration of apocynin, catalase, and a combination of apocynin and catalase improved (P<0.05) the fractional excretion of sodium and urinary sodium to potassium ratio in CsA especially by day 14. A similar pattern of results was seen for urinary protein excretion in CsA rats treated with apocynin, catalase, and their combination in that there was a decrease (all P<0.05) in urinary protein levels to baseline values (day 0) as early as 7 days after initiation of the treatment. Moreover, individual or combined treatment of CsA rats with apocynin and catalase resulted in near control urine protein values in these rats as demonstrated in Fig 7.

Fig 7 — CsA, Cyclosporine A. Data presented as mean±SEM. * p<0.05 from Day 0; # p<0.05 of Cx vs. C (Day 14); † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx (Day 14).

There was a significant increase (P<0.05) in kidney index in CsA rats as compared to control rats as shown in Fig 8. However, treatment of CsA rats for 14 days with apocynin, catalase, and their combination decreased (P<0.05) kidney index compared to untreated CsA rats. No changes in kidney index were observed in control rats treated with apocynin, catalase, and a combination of apocynin and catalase.

The BUN values for treatment groups at day 16 of the study are shown in Fig 9. The BUN in CsA group was significantly higher (P<0.05) as compared to control group. However, CsA rats treated with apocynin, catalase, and especially their combination showed significantly lower BUN compared to untreated CsA. BUN levels in control rats treated with apocynin, catalase or their combination were not different from their untreated counterparts.

Apocynin and catalase treatment restored blood pressure and heart rate in CsA rats

Baseline SBP and heart rate were similar in the eight experimental groups. SBP was significantly (P<0.05) higher than baseline levels in CsA rats at day 7 and day 14 of the treatment period. Similarly, heart rate was also increased significantly (P<0.05) from baseline in CsA and CsA rats treated with catalase starting at day 7 and continued until the end of the 14-day study period. Treatment of CsA rats with apocynin, catalase or a combination of apocynin and catalase significantly attenuated the increase in both SBP and heart rate as shown in Figs 10 and 11.

Fig 10 — CsA, Cyclosporine A. Data presented as mean±SEM. * p<0.05 from Day 0; # p<0.05 of Cx vs. C (Day 14); ¥ p<0.05 of all groups vs. C except Cx (Day 14); † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx (Day 14).

Fig 11 — CsA, Cyclosporine A. Data presented as mean±SEM. * p<0.05 from Day 0; # p<0.05 of Cx vs. C (Day 14); ¥ p<0.05 of all groups vs. C except Cx (Day 14); † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx (Day 14).

Apocynin and catalase restored renal and systemic hemodynamic parameters in CsA rats

The basal values of RCBP in CsA rats were significantly lower (P<0.05) as compared to control rats. However, treatment of CsA rats with apocynin, catalase, and especially their combination inhibited this decrease in RCBP compared to untreated CsA rats. No differences were observed in RCBP between control rats treated with apocynin, catalase or a combination of apocynin and catalase (Fig 12).

Fig 12 — CsA, Cyclosporine A. Data presented as mean±SEM. # p<0.05 of Cx vs. C; † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx; ¶ p<0.05 of CxCat vs. CxApoCat.

PWV is used clinically as a parameter of aortic stiffness and an established marker of cardiovascular risk. In this study, CsA rats had a higher (P<0.05) PWV at the end of the study period compared to their control counterparts. However, treatment of CsA rats with apocynin, catalase or a combination of apocynin and catalase restored the PWV values to near normal values. No significant differences were observed in PWV values in untreated control rats or those treated with either apocynin, catalase or a combination of apocynin and catalase (Fig 13).

Fig 13 — CsA, Cyclosporine A. Data presented as mean±SEM. # p<0.05 of Cx vs. C; † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx.

β-index is used to quantify the intrinsic exponent of the blood pressure and vascular diameter relationship. Unlike the other treatment groups, CsA treated rats had a significantly higher (P<0.05) β-index. Administration of apocynin, catalase or a combination of apocynin and catalase restored the β-index in CsA treated rats to values comparable to control (Fig 14).

Fig 14 — CsA, Cyclosporine A. Data presented as mean±SEM. # p<0.05 of Cx vs. C; † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx.

Apocynin and catalase inhibited the increase in oxidative stress markers due to CsA

The plasma and kidney levels of malondialdehyde (MDA) are depicted in Figs 15 and 16, respectively. CsA rats have higher (P<0.05) plasma MDA levels compared to control rats. However, treatment of CsA rats with apocynin, catalase or a combination of apocynin and catalase restored MDA levels to near normal levels. However, plasma MDA in catalase treated CsA rats was higher (P<0.05) compared to CsA rats treated with a combination of apocynin and catalase. No significant changes in plasma MDA levels were observed in untreated or apocynin, catalase or apocynin plus catalase treated control rats (Fig 15). The kidney level of MDA in CsA rats was higher (P<0.05) than control rats. However, treatment of CsA rats with apocynin, catalase or a combination of apocynin and catalase resulted in decreased (P<0.05) kidney MDA levels compared to untreated CsA rats (Fig 16).

Fig 15 — CsA, Cyclosporine A. Values are mean±SEM. ¥ p<0.05 of all group vs. C except Cx; # p<0.05 of Cx vs. C; † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx; § p<0.05 of CxApo vs. CxApoCat; ¶ p<0.05 of CxCat vs. CxApoCat; δ p<0.05 of CxCat vs. CxApo.

Fig 16 — CsA, Cyclosporine A. Values are mean±SEM. ¥ p<0.05 of all group vs. C except Cx; # p<0.05 of Cx versus C; and † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx.

The activity of SOD in the plasma and kidney of experimental groups are shown in Figs 17 and 18, respectively. CsA rats had a significantly lower (P<0.05) plasma SOD activity as compared to control. The administration of apocynin in combination with catalase, or either drug alone, in the CsA rat resulted in higher (P<0.05) SOD activity compared to untreated rats. In addition, there was a significantly higher plasma SOD activity level in control rats treated with apocynin or apocynin plus catalase compared to untreated controls (Fig 17). SOD activity in the kidney was lower (P<0.05) in CsA rats compared to control but was similar to control levels in CsA rats treated with apocynin, catalase or a combination of apocynin and catalase (Fig 18).

Fig 17 — CsA, Cyclosporine A. Values are mean±SEM. ¥ p<0.05 of all group vs. C except Cx; # p<0.05 of Cx vs. C; † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx; § p<0.05 of CxApo vs. CxApoCat; ¶ p<0.05 of CxCat vs. CxApoCat; δ p<0.05 of CxCat vs. CxApo.

Fig 18 — CsA, Cyclosporine A. Values are mean±SEM. ¥ p<0.05 of all group vs. C except Cx; # p<0.05 of Cx vs. C; and † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx.

Plasma nitric oxide (NO) activity in the different treatment groups is illustrated in Fig 19. The plasma NO activity in CsA rats was lower (P<0.05) compared to control rats. Treatment of CsA rats with apocynin, catalase, and their combination restored NO activity back towards normal levels. The plasma NO activity in control rats treated with apocynin and apocynin plus catalase was significantly higher (P<0.05) than untreated control rats.

The plasma levels of T-AOC in CsA rats were lower (P<0.05) than their control counterparts. However, the levels of T-AOC in CsA rats treated with apocynin, catalase, and the combination of apocynin and catalase were higher compared to the rats without therapy (Fig 20). On another hand, the plasma levels of T-AOC in control rats treated with apocynin and apocynin plus catalase were higher than untreated controls. The kidney levels of T-AOC in CsA rats were also lower (P<0.05) compared to their control counterparts. Administration of apocynin, catalase, and a combination of apocynin and catalase resulted in higher kidney tissue T-AOC (P<0.05) compared to untreated CsA rats Fig 21.

Fig 20 — CsA, Cyclosporine A. Values are mean±SEM. ¥ p<0.05 of all group vs. C except Cx; # p<0.05 of Cx vs. C; † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx.

Fig 21 — CsA, Cyclosporine A. Values are mean±SEM. # p<0.05 of Cx vs. C; and † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx.

Apocynin and catalase restored renal Nox4 mRNA expression in CsA rats

Nox4 mRNA expression in CsA group was higher, by some 64% (P<0.05), compared to control group. However, treatment of CsA rats with apocynin alone resulted in similar Nox4 expression to control rats treated with apocynin. Likewise, the level of Nox4 mRNA expression in CsA rats treated with catalase alone was comparable to untreated controls. The treatment of CsA rats with a combination of catalase and apocynin resulted in lower Nox4 mRNA expression when compared to untreated CsA rats (all P<0.05) as shown in Fig 22. The level of kidney Nox4 mRNA expression was lower by almost 35% (P<0.05) in apocynin treated compared to untreated control rats. A similar observation was seen in control rats treated with a combination of apocynin and catalase where Nox4 expression was lower than untreated rats.

Fig 22 — CsA, Cyclosporine A. Values are mean±SEM. ¥ p<0.05 of all group vs. C except Cx; # p<0.05 of Cx vs. C; † p<0.05 of CxApo, CxCat and CxApoCat vs. Cx; § p<0.05 of CxApo vs. CxApoCat; ¶ p<0.05 of CxCat vs. CxApoCat; δ p<0.05 of CxCat vs. CxApo.

Apocynin and catalase prevented structural alterations due to CsA-induced tissue damage

The kidney tissues did not show any ultra-structural alterations in the glomerulus and tubules components in control rats treated or not with apocynin, catalase, and a combination of apocynin and catalase as demonstrated in Fig 23A, 23B, 23C and 23D. The kidney tissues of CsA rats showed severe renal tubular ischaemia (Fig 23E2, solid arrow) as well as abscesses in the renal interstitium area. Neutrophils surrounded by foamy macrophages were also observed beyond the subcapsular region (Fig 23E1, 23E2 & 23E3, open arrow). A deformed glomerular apparatus characterized by thickening of Bowmen’s capsules was also observed in CsA rats (Fig 23E1, solid arrow). There was also a possible striped interstitial fibrosis (Fig 23E3, solid arrow). All structural changes in the kidney of CsA rats were not detected following apocynin, catalase or combined apocynin and catalase treatment (Fig 23F, 23G & 23H respectively).

Fig 23 — Arrows indicate the area of the renal tissue with histological changes. CsA, Cyclosporine A. (Haematoxylin and eosin stain; original magnification x100).

Discussion

The present study examined the combined effect of NADPH oxidase inhibition using apocynin and H₂O₂ scavenging using catalase on CsA-induced renal injury. CsA rats in the present study demonstrated hypertension, impaired renal function and elevated oxidative stress. Moreover, histopathological examination of CsA rat kidney showed inflammatory cells infiltration and interstitial fibrosis. Therefore, CsA induces an oxidative stress cascade with negative impacts on both renal excretory function and renal histology leading to hypertension. The novel finding of this study is that treatment of CsA renal injury rats with a combination of apocynin and catalase for 14 days ameliorated hypertension, renal dysfunction and oxidative stress.

The administration of CsA for 14 days in this study impaired renal function as manifested by a decrease in creatinine clearance and urinary sodium excretion as well as kidney tissue damage as shown by histology. There was a significant decrease in body weight of CsA treated rats which began on day 7 of the study and onward. Morphake et al., [39] showed using CsA model in rats a decrease in creatinine clearance in addition to body weight loss by almost 13% within 7 days of CsA treatment. This magnitude is comparable to the loss in body weight in the present study after 7 days. The reason of this loss is possibly related to anorexic effect of CsA and in agreement with previous studies in rats [39–41]. Weight loss can also be attributed to the higher metabolic rate in response to the catabolic action of CsA [42]. In addition to body weight loss, CsA treatment was associated with a significant increase in blood pressure as early as 7 days of the study. Likewise, there was an increase in heart rate which is possibly related to augmented sympathetic nerve activity in this model [43]. In relation to that, an activation of renal afferent nerve activity was reported before that led to a reflex sympathoexcitation and increased blood pressure and heart rate [44, 45]. The treatment of CsA rats with a combination of apocynin and catalase in this study ameliorated the body weight loss and restored blood pressure and heart rate values towards normal. Indeed, treatment with either apocynin or catalase was shown to have an antihypertensive effect in this study as well as previous studies [46, 47].

The present study showed a reduction by more than 50% in the fractional excretion of sodium (FE_Na+) from its baseline value (day 0) after 14 days of CsA treatment. This effect of CsA on sodium reabsorption has been suggested to contribute to the increase in blood pressure in this model [48]. The decrease in sodium excretion in CsA rats in this study is related to increased expression of epithelial sodium channels (ENaC) in the nephron which was previously reported in CsA treated allogeneic rat model [49]. Moreover, in spontaneously hypertensive rat (SHR) which is a model of renal injury and hypertension there is enhanced expression of ENaC with possible role in sodium retention observed during the course of hypertension in this model [50]. Impaired sodium excretion in CsA model can also be due to O₂⁻induced activation of protein kinase C [51] and increased activity of apical membrane Na⁺-K⁺-2Cl^- co-transporter in the thick ascending limb of the loop of Henle [52]. Based on these studies, the mechanism of decreased sodium excretion in CsA model can be multifaceted.

The CsA rats in this study also showed a decrease in urinary sodium to potassium ratio after 7 days of CsA treatment initiation. This finding points to a possible involvement of renin-angiotensin aldosterone system in CsA-induced hypertension in this model. This notion is supported by a recent study using CsA in rats which showed that aldosterone is a key mediator of renal injury in this model an effect which can be inhibited by aldosterone antagonists [34]. Indeed, apocynin treatment of CsA rats in this study restored the sodium to potassium ratio. A previous study showed that apocynin inhibits aldosterone/salt-induced kidney damage by preventing the production of prostaglandin E₂ (PGE₂) in Dahl salt-sensitive rat model [53]. Furthermore, an inverse relationship was reported between baseline 24-h urinary sodium to potassium ratio and the 24-h urinary potassium excretion especially during diuresis in SHR model which is characterised by sympathetic hyperactivity [25, 54]. The effects of apocynin and catalase in the present study on urinary sodium to potassium ratio could possibly be linked to their inhibitory effect on ROS and consequently on sympathetic hyperactivity [55]. CsA treatment in this study was associated with increased urinary protein excretion and structural alterations such as deformation of glomerular apparatus, tubular ischemia and the presence of inflammatory exudates in the subcapsular region. The combined administration of apocynin and catalase in this study exerted a protective effect against CsA-induced kidney injury due to O₂^- and H₂O₂ removal from tissues by these compounds respectively [56, 57].

The CsA-induced renal injury in this study was accompanied by a decrease in renal cortical blood perfusion. A previous study by Prevot et al, [58] showed a relationship between decreased renal blood flow in this model and local vasoconstrictive mechanism in the kidney. Several mechanisms could be involved such as activation of the renin-angiotensin system, imbalances in prostaglandins, thromboxane and hypersecretion of endothelin-1 can also play a role in CsA-induced renal vasoconstriction [1]. Treatment of CsA rats with apocynin and catalase in the present study increased renal cortical blood perfusion compared to CsA rats without treatment. The combined treatment with apocynin and catalase had a greater effect on renal blood perfusion compared to individual treatment. This observation suggests that concomitant NADPH oxidase inhibition and H₂O₂ hydrolysis might be required to maximise the antioxidant effect. This can be looked at in the context of the classical oxidative pathway where O₂^- is converted into H₂O₂ by SOD enzyme which will be eventually converted into H₂O and molecular O₂. However, a heightened O₂^- dismutation alone in the absence of concomitant increase in catalase activity would result in oxidative stress mediated by H₂O₂ abundance. Therefore, direct NADPH oxidase inhibition in addition to continuous hydrolysis of H₂O₂ using apocynin and catalase combined treatment can provide a better oxidative defence against CsA-induced renal injury.

The plasma and kidney MDA levels in this study were significantly higher in CsA rats compared to controls. In relation to that, it was suggested that CsA impairs mitochondrial electron transport and cytochrome P450-3A activity leading to oxidative stress [59]. Regardless of the source of free radicals, intervention that blocks this pathway may be an effective strategy to prevent ROS-mediated kidney damage. The combined treatment of CsA rats with apocynin and catalase in this study restored MDA levels to near normal values both locally and in the plasma.

The pulse wave velocity parameters were significantly impaired in CsA rats in the present study. Indeed, oxidative stress is well studied as a cause of altered arterial elasticity [60]. This finding is consonant with a report on the acute effect of CsA on haemodynamics in the large arteries in kidney transplant patients [61]. Treatment of CsA rats with a combination of apocynin and catalase restored pulse wave velocity and improved the β-index possibly due to up-regulation of antioxidant enzymes that drive the scavenging activity of NADPH oxidase. This reduces ROS production and speeds up the conversion of H₂O₂ to O₂ and H₂O [62] with effects on haemodynamics.

NOX4 is readily distinguished from other NADPH oxidase isoforms in the NOX family of proteins by its high level of expression in renal tissues [63]. It was evident that, within the renal cortex, NADPH oxidase activity was significantly elevated in rats subjected to CsA [46]. The findings of the present study demonstrated that CsA administration increases NOX4 expression in the renal homogenates and it is thought to be consistent with its involvement in the generation of O₂^-. In line with these findings, previous investigations showed elevated expression of NOX4 in a number of cardiovascular diseases including atherosclerosis, hypertension, cardiac failure and ischemic stroke [64]. Treatment of CsA rats with a combination of apocynin and catalase in this study decreased NOX4 mRNA expression to a greater extent compared to individual treatment with either apocynin or catalase alone. This could be related to the crosstalk between direct O₂^- elimination by apocynin and continuous H₂O₂ hydrolysis by catalase. It has also been suggested that apocynin and catalase prevent translocation of the cytosolic components of NOX, p47^phox to gp91^phox required to form the active oxidase [65]. Both apocynin and catalase exerted the same degree of protection against Dahl salt sensitive rat hypertension model in a previous study [66]. The findings of this study further demonstrate a stronger effect due to combined treatment with apocynin and catalase in ameliorating oxidative stress in CsA model of renal injury.

Conclusion

Our findings suggest that renal injury in CsA model was associated with an increase in NADPH oxidase activity, which contributes to the production of O₂^- and to the development of renal injury and hypertension. The prophylactic treatment with apocynin and catalase for 14 days maintained normal blood pressure and improved renal function which eventually halted CsA-induced tissue damage. Based on renal functional and oxidative stress data, the current study demonstrated that apocynin provides more defence against oxidative stress, impaired kidney function and hypertension compared with catalase. Combined treatment with apocynin and catalase had a greater effect compared to individual drug in restoring blood pressure and renal function. Taken together, inhibiting NADPH oxidase by apocynin, catalase, and their combination prevents the onset of hypertension and renal injury induced by CsA.

Acknowledgments

We thank the Pantai Premier Pathology Sdn Bhd for professional service and advice on histopathology studies.

Data Availability

All relevant data are within the paper.

Funding Statement

Tan Yong Chia; will be the corresponding author for this paper. This study was funded by the Research University grant from University Sains Malaysia number 1001/PFARMASI/811347. Tan Yong Chia is a recipient of a research fellowship from the Institute of Postgraduate Studies (IPS) of University Sains Malaysia. NO-Include this sentence at the end of your statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Yoon HE, Yang CW. Established and newly proposed mechanisms of chronic cyclosporine nephropathy. The Korean journal of internal medicine. 2009;24(2):81 10.3904/kjim.2009.24.2.81 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Ojo AO, Held PJ, Port FK, Wolfe RA, Leichtman AB, Young EW, et al. Chronic renal failure after transplantation of a nonrenal organ. New England Journal of Medicine. 2003;349(10):931–40. 10.1056/NEJMoa021744 [DOI] [PubMed] [Google Scholar]
3.Colgan J, Asmal M, Yu B, Luban J. Cyclophilin A-deficient mice are resistant to immunosuppression by cyclosporine. The Journal of Immunology. 2005;174(10):6030–8. 10.4049/jimmunol.174.10.6030 [DOI] [PubMed] [Google Scholar]
4.Friedman J, Weissman I. Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA. Cell. 1991;66(4):799–6. 10.1016/0092-8674(91)90123-g [DOI] [PubMed] [Google Scholar]
5.Bobadilla NA, Gamba G. New insights into the pathophysiology of cyclosporine nephrotoxicity: a role of aldosterone. American Journal of Physiology-Renal Physiology. 2007;293(1):F2–F9. 10.1152/ajprenal.00072.2007 [DOI] [PubMed] [Google Scholar]
6.Heering P, Schadewaldt P, Bach D, Grabensee B. Nephrotoxicity of cyclosporine in humans: effect of cyclosporine on glomerular filtration and proximal tubular reabsorption. The clinical investigator. 1993;71(12):1010–5. 10.1007/bf00180033 [DOI] [PubMed] [Google Scholar]
7.Smith SR, Creech EA, Schaffer AV, Martin LL, Rakhit A, Douglas FL, et al. Effects of thromboxane synthase inhibition with CGS 13080 in human cyclosporine nephrotoxicity. Kidney international. 1992;41(1):199–5. 10.1038/ki.1992.27 [DOI] [PubMed] [Google Scholar]
8.Burdmann EA, Andoh TF, Yu L, Bennett WM. Cyclosporine nephrotoxicity. Seminars in nephrology. 2003;23:465–76. 10.1016/s0270-9295(03)00090-1 [DOI] [PubMed] [Google Scholar]
9.Chia TY, Sattar MA, Abdulla MH, Rathore HA, Ahmad FuD, Kaur G, et al. The effects of tempol on renal function and hemodynamics in cyclosporine-induced renal insufficiency rats. Renal failure. 2013;35(7):978–88. 10.3109/0886022X.2013.809563 [DOI] [PubMed] [Google Scholar]
10.Ciresi DL, Lloyd MA, Sandberg SM, Heublein DM, Edwards BS. The sodium retaining effects of cyclosporine. Kidney international. 1992;41(6):1599–605. 10.1038/ki.1992.231 [DOI] [PubMed] [Google Scholar]
11.Pichler RH, Franceschini N, Young BA, Hugo C, Andoh TF, Burdmann EA, et al. Pathogenesis of cyclosporine nephropathy: roles of angiotensin II and osteopontin. Journal of the American Society of Nephrology. 1995;6(4):1186–96. [DOI] [PubMed] [Google Scholar]
12.Modlinger P, Chabrashvili T, Gill PS, Mendonca M, Harrison DG, Griendling KK, et al. RNA silencing in vivo reveals role of p22phox in rat angiotensin slow pressor response. Hypertension. 2006;47(2):238–44. 10.1161/01.HYP.0000200023.02195.73 [DOI] [PubMed] [Google Scholar]
13.De Keulenaer WG, Alexander RW, Ushio-Fukai M, Ishizaka N, Griendling KK. Tumour necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle. Biochemical journal. 1998;329(3):653–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Brandes RP, Viedt C, Nguyen K, Beer S, Kreuzer J, Busse R, et al. Thrombin-induced MCP-1 expression involves activation of the p22phox-containing NADPH oxidase in human vascular smooth muscle cells. Thrombosis and haemostasis. 2001;85(06):1104–10. [PubMed] [Google Scholar]
15.Lassegue B, Clempus RE. Vascular NAD (P) H oxidases: specific features, expression, and regulation. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 2003;285(2):R277–R97. 10.1152/ajpregu.00758.2002 [DOI] [PubMed] [Google Scholar]
16.Guzik TJ, Chen W, Gongora MC, Guzik B, Lob HE, Mangalat D, et al. Calcium-dependent NOX5 nicotinamide adenine dinucleotide phosphate oxidase contributes to vascular oxidative stress in human coronary artery disease. Journal of the American College of Cardiology. 2008;52(22):1803–9. 10.1016/j.jacc.2008.07.063 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Guzik TJ, Sadowski J, Guzik B, Jopek A, Kapelak B, Przybylowski P, et al. Coronary artery superoxide production and nox isoform expression in human coronary artery disease. Arteriosclerosis, thrombosis, and vascular biology. 2006;26(2):333–9. 10.1161/01.ATV.0000196651.64776.51 [DOI] [PubMed] [Google Scholar]
18.Banfi B, Tirone F, Durussel I, Knisz J, Moskwa P, Molnar GZ, et al. Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). Journal of Biological Chemistry. 2004;279(18):18583–91. 10.1074/jbc.M310268200 [DOI] [PubMed] [Google Scholar]
19.Jha JC, Banal C, Okabe J, Gray SP, Hettige T, Chow BSM, et al. NADPH oxidase Nox5 accelerates renal injury in diabetic nephropathy. Diabetes. 2017;66(10):2691–703. 10.2337/db16-1585 [DOI] [PubMed] [Google Scholar]
20.Cucoranu I, Clempus R, Dikalova A, Phelan PJ, Ariyan S, Dikalov S, et al. NAD (P) H oxidase 4 mediates transforming growth factor-Beta1-induced differentiation of cardiac fibroblasts into myofibroblasts. Circulation research. 2005;97(9):900–7. 10.1161/01.RES.0000187457.24338.3D [DOI] [PubMed] [Google Scholar]
21.Chen F, Haigh S, Barman SA, Fulton D. From form to function: the role of Nox4 in the cardiovascular system. Frontiers in physiology. 2012;3:412 10.3389/fphys.2012.00412 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Parfenova H. Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF {alpha} in cerebral vascular endothelial cells Am J Physiol Cell Physiol 2009;296:C422–C32. 10.1152/ajpcell.00381.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Yang X, Zhao K, Deng W, Zhao L, Jin H, Mei F, et al. Apocynin Attenuates Acute Kidney Injury and Inflammation in Rats with Acute Hypertriglyceridemic Pancreatitis. Digestive Diseases and Sciences. 2019:1–13. [DOI] [PubMed] [Google Scholar]
24.Chia TY, Sattar MA, Abdullah MH, Ahmad FUD, Ibraheem ZO, Lia KJ, et al. Cyclosporine A—induced nephrotoxic Sprague-Dawley rats are more suscetable to altered vascular function and haemodynamic. International journal of pharmacy and pharmaceutical sciences. 2012;4:431–9. [Google Scholar]
25.Ahmad FUD, Sattar MA, Rathore HA, Tan YC, Akhtar S, Jin OH, et al. Hydrogen sulphide and tempol treatments improve the blood pressure and renal excretory responses in spontaneously hypertensive rats. Renal failure. 2014;36(4):598–5. 10.3109/0886022X.2014.882218 [DOI] [PubMed] [Google Scholar]
26.Ahmad FUD, Sattar MA, Rathore HA, Hussain AI, Chia TY, Jin OH, et al. Exogenous hydrogen sulfide attenuates oxidative stress in spontaneously hypertensive rats. International Journal of Pharmaceutical Sciences and Research. 2013;4(8):2916. [Google Scholar]
27.Ahmad A, Sattar MA, Azam M, Khan SA, Bhatt O, Johns EJ. Interaction between nitric oxide and renal alpha1-adrenoreceptors mediated vasoconstriction in rats with left ventricular hypertrophyin Wistar Kyoto rats. PloS one. 2018;13(2):e0189386 10.1371/journal.pone.0189386 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Swarup KRLA, Sattar MA, Abdullah NA, Abdulla MH, Salman IM, Rathore HA, et al. Effect of dragon fruit extract on oxidative stress and aortic stiffness in streptozotocin-induced diabetes in rats. Pharmacognosy research. 2010;2(1):31–5. 10.4103/0974-8490.60582 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Vlachopoulos C, Aznaouridis K, Terentes-Printzios D, Ioakeimidis N, Stefanadis C. Prediction of cardiovascular events and all-cause mortality with brachial-ankle elasticity index: a systematic review and meta-analysis. Hypertension. 2012;60(2):556–62. 10.1161/HYPERTENSIONAHA.112.194779 [DOI] [PubMed] [Google Scholar]
30.Wang Y-X, Halks-Miller M, Vergona R, Sullivan ME, Fitch R, Mallari C, et al. Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice. American Journal of Physiology-Heart and Circulatory Physiology. 2000;278(2):H428–H34. 10.1152/ajpheart.2000.278.2.H428 [DOI] [PubMed] [Google Scholar]
31.Cosson E, Herisse M, Laude D, Thomas F, Valensi PE, Attali J-R, et al. Aortic stiffness and pulse pressure amplification in Wistar-Kyoto and spontaneously hypertensive rats. American Journal of Physiology-Heart and Circulatory Physiology. 2007; 292(5):H2506–12 10.1152/ajpheart.00732.2006 [DOI] [PubMed] [Google Scholar]
32.Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical biochemistry. 1979;95(2):351–8. 10.1016/0003-2697(79)90738-3 [DOI] [PubMed] [Google Scholar]
33.Oyanagui Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Analytical biochemistry. 1984;142(2):290–6. 10.1016/0003-2697(84)90467-6 [DOI] [PubMed] [Google Scholar]
34.Archer S. Measurement of nitric oxide in biological models. The FASEB journal. 1993;7(2):349–60. 10.1096/fasebj.7.2.8440411 [DOI] [PubMed] [Google Scholar]
35.Sun J, Zhang X, Broderick M, Fein H. Measurement of nitric oxide production in biological systems by using Griess reaction assay. Sensors. 2003;3(8):276–84. [Google Scholar]
36.Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clinical science. 1993;84(4):407–12. 10.1042/cs0840407 [DOI] [PubMed] [Google Scholar]
37.Kitiyakara C, Chabrashvili T, Chen Y, Blau J, Karber A, Aslam S, et al. Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase. Journal of the American Society of Nephrology. 2003;14(11):2775–82. 10.1097/01.asn.0000092145.90389.65 [DOI] [PubMed] [Google Scholar]
38.Ahmad A, Sattar MA, Rathore HA, Abdulla MH, Khan SA, Azam M, et al. Up Regulation of cystathione y lyase and Hydrogen Sulphide in the Myocardium Inhibits the Progression of Isoproterenol-Caffeine Induced Left Ventricular Hypertrophy in Wistar Kyoto Rats. PloS one. 2016;11(3):e0150137 10.1371/journal.pone.0150137 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Morphake P, Bariety J, Darlametsos I, Tsipas G, Gkikas G, Hornysh A, et al. Alteration of cyclosporine (CsA)-induced nephrotoxicity by gamma linolenic acid (GLA) and eicosapentaenoic acid (EPA) in wistar rats. Prostaglandins, leukotrienes and essential fatty acids. 1994;50(1):29–35. [DOI] [PubMed] [Google Scholar]
40.Chia TY, Sattar MA, Rathore HA, Abdullah MH, Singh GKC, Ahmad FUD, et al. The effects of tempol on renal function and hemodynamics in cyclosporine-induced renal insufficiency rats. Renal failure. 2013;35(7):978–88. 10.3109/0886022X.2013.809563 [DOI] [PubMed] [Google Scholar]
41.Fassi A, Sangalli F, Colombi F, Perico N, Remuzzi G, Remuzzi A. Beneficial effects of calcium channel blockade on acute glomerular hemodynamic changes induced by cyclosporine. American journal of kidney diseases. 1999;33(2):267–75. 10.1016/s0272-6386(99)70299-4 [DOI] [PubMed] [Google Scholar]
42.Hagar HH, El Etter E, Arafa M. Taurine attenuates hypertension and renal dysfunction induced by cyclosporine A in rats. Clinical and experimental pharmacology and physiology. 2006;33(3):189–96. 10.1111/j.1440-1681.2006.04345.x [DOI] [PubMed] [Google Scholar]
43.Naesens M, Kuypers DRJ, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clinical Journal of the American Society of Nephrology. 2009;4(2):481–8. 10.2215/CJN.04800908 [DOI] [PubMed] [Google Scholar]
44.Campese VM, Kogosov E. Renal afferent denervation prevents hypertension in rats with chronic renal failure. Hypertension. 1995;25(4):878–82. [DOI] [PubMed] [Google Scholar]
45.Hausberg M, Kosch M, Harmelink P, Barenbrock M, Hohage H, Kisters K, et al. Sympathetic nerve activity in end-stage renal disease. Circulation. 2002;106(15):1974–9. 10.1161/01.cir.0000034043.16664.96 [DOI] [PubMed] [Google Scholar]
46.Ciarcia R, Damiano S, Florio A, Spagnuolo M, Zacchia E, Squillacioti C, et al. The protective effect of apocynin on cyclosporine A-induced hypertension and nephrotoxicity in rats. Journal of cellular biochemistry. 2015;116(9):1848–56. 10.1002/jcb.25140 [DOI] [PubMed] [Google Scholar]
47.Sousa T, Oliveira S, Afonso J, Morato M, Patinha D, Fraga S, et al. Role of H₂O₂ in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II. British journal of pharmacology. 2012;166(8):2386–401. 10.1111/j.1476-5381.2012.01957.x [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Hoorn EJ, Walsh SB, McCormick JA, Zietse R, Unwin RJ, Ellison DH. Pathogenesis of calcineurin inhibitor-induced hypertension. Journal of Nephrology. 2012;25(3):269–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Edemir B, Reuter S, Borgulya R, Schroter R, Neugebauer U, Gabriels G, et al. Acute rejection modulates gene expression in the collecting duct. Journal of the American Society of Nephrology. 2008;19(3):538–46. 10.1681/ASN.2007040513 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Kim SW, Wang W, Kwon T-H, Knepper MA, Frokiaer J, Nielsen S. Increased expression of ENaC subunits and increased apical targeting of AQP2 in the kidneys of spontaneously hypertensive rats. American Journal of Physiology-Renal Physiology. 2005; 289: F957–F68. 10.1152/ajprenal.00413.2004 [DOI] [PubMed] [Google Scholar]
51.Ortiz PA, Garvin JL. Superoxide stimulates NaCl absorption by the thick ascending limb. American Journal of Physiology-Renal Physiology. 2002;283(5):F957–F62. 10.1152/ajprenal.00102.2002 [DOI] [PubMed] [Google Scholar]
52.Juncos R, Garvin JL. Superoxide enhances Na-K-2Cl cotransporter activity in the thick ascending limb. American Journal of Physiology-Renal Physiology. 2005;288(5):F982–F7. 10.1152/ajprenal.00348.2004 [DOI] [PubMed] [Google Scholar]
53.Bayorh MA, Rollins-Hairston A, Adiyiah J, Lyn D, Eatman D. Eplerenone inhibits aldosterone-induced renal expression of cyclooxygenase. Journal of the Renin-Angiotensin-Aldosterone System. 2012;13(3):353–9. 10.1177/1470320312443911 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Eudy RJ, Sahasrabudhe V, Sweeney K, Tugnait M, King-Ahmad A, Near K, et al. The use of plasma aldosterone and urinary sodium to potassium ratio as translatable quantitative biomarkers of mineralocorticoid receptor antagonism. Journal of translational medicine. 2011;9(1):180. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Zhang Z-H, Yu Y, Wei S-G, Felder RB. Centrally administered lipopolysaccharide elicits sympathetic excitation via NAD (P) H oxidase-dependent mitogen-activated protein kinase signaling. Journal of hypertension. 2010;28(4):806 10.1097/HJH.0b013e3283358b6e [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Altintas R, Polat A, Vardi N, Oguz F, Beytur A, Sagir M, et al. The protective effects of apocynin on kidney damage caused by renal ischemia/reperfusion. Journal of endourology. 2013;27(5):617–24. 10.1089/end.2012.0556 [DOI] [PubMed] [Google Scholar]
57.Ma SF, Nishikawa M, Hyoudou K, Takahashi R, Ikemura M, Kobayashi Y, et al. Combining cisplatin with cationized catalase decreases nephrotoxicity while improving antitumor activity. Kidney international. 2007;72(12):1474–82. 10.1038/sj.ki.5002556 [DOI] [PubMed] [Google Scholar]
58.Prevot A, Semama DS, Justrabo E, Guignard JP, Escousse A, Gouyon JB. Acute cyclosporine A-induced nephrotoxicity: a rabbit model. Pediatric nephrology. 2000;14(5):370–5. 10.1007/s004670050777 [DOI] [PubMed] [Google Scholar]
59.Nishiyama A, Kobori H, Fukui T, Zhang G-X, Yao L, Rahman M, et al. Role of angiotensin II and reactive oxygen species in cyclosporine A-dependent hypertension. Hypertension. 2003;42(4):754–60. 10.1161/01.HYP.0000085195.38870.44 [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Patel RS, Al Mheid I, Morris AA, Ahmed Y, Kavtaradze N, Ali S, et al. Oxidative stress is associated with impaired arterial elasticity. Atherosclerosis. 2011;218(1):90–5. 10.1016/j.atherosclerosis.2011.04.033 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Covic A, Mardare N, Gusbeth-Tatomir P, Buhaescu I, Goldsmith DJA. Acute effect of CyA A (Neoral (R)) on large artery hemodynamics in renal transplant patients. Kidney international. 2005;67(2):732–7. 10.1111/j.1523-1755.2005.67134.x [DOI] [PubMed] [Google Scholar]
62.Hamilton CA, Miller WH, Sammy A-B, Brosnan MJ, Drummond RD, McBride MW, et al. Strategies to reduce oxidative stress in cardiovascular disease. Clinical science. 2004;106(3):219–34. 10.1042/CS20030379 [DOI] [PubMed] [Google Scholar]
63.Griendling KK. NADPH oxidases: new regulators of old functions. Antioxidants & redox signaling. 2006;8(9–10):1443–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Griendling KK. Novel NAD (P) H oxidases in the cardiovascular system. Heart. 2004;90(5):491–3. 10.1136/hrt.2003.029397 [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Touyz RM. Apocynin, NADPH oxidase, and vascular cells: a complex matter. Hypertension. 2008; 51(2):172–4. 10.1161/HYPERTENSIONAHA.107.103200 [DOI] [PubMed] [Google Scholar]
66.Taylor NE, Glocka P, Liang M, Cowley AW Jr. NADPH oxidase in the renal medulla causes oxidative stress and contributes to salt-sensitive hypertension in Dahl S rats. Hypertension. 2006;47(4):692–8. 10.1161/01.HYP.0000203161.02046.8d [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0231472.r001

Decision Letter 0

Partha Mukhopadhyay

17 Oct 2019

PONE-D-19-26893

Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats

PLOS ONE

Dear Mr TAN SAMUAL,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your manuscript was reviewed by two experts and both of them raised major points, which require your attention.

We would appreciate receiving your revised manuscript by Dec 01 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Partha Mukhopadhyay, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study evaluated the effect of apocymin and catalase in cyclosporine A-induced nephrotoxicity. Several biomarkers of oxidative stress were determined due to oxidative stress participated in nephrotoxicity as an important pathogenesis mechanism. The authors tested the MDA, SOD, NO and AOC level in plasma from different groups. Why do the authors test these biomarkers in the kidney tissue since the authors the major tissue of injury induced by cyclosporine is the kidney tissue?

The renal function parameters including serum Cr level, blood urea nitrogen (BUN) level, creatinine clearance, proteinuria directly reflected the kidney function and damage condition. BUN level is an important indicator of kidney function. Did the authors test the BUN level in different groups?

Apocynin can significantly inhibit the activity of NOXes. The authors evaluated the NOX4 mRNA expression in kidney tissue from different groups. Could the author show the NOX4 protein expression in the kidney tissue using IF or IHC?

Reviewer #2: In the study, ' Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats', the authors have demonstrated a potential therapeutic role for the agents combined, in treating harmful side effects associated with CsA treatment. While the study was designed and conducted well, there were some issues as highlighted below:

1) Abstract: Please rewrite as it is muddled. Do not abbreviate terms (CsA) without first mentioning the proper name. The abstract launches into the experimental design without any background (can be brief) and rationale. Please incorporate these, and structure properly. Also, in describing experimental results, mention the effects of apocynin and catalase alone, and then emphasize that the combinatorial treatment was more potent.

2) Introduction: The authors say that CsA is a widely used immunosuppressant in organ transplantation. What is the rate of occurrence of nephrotoxicity in these patients clinically?

3)Introduction: Mention that IL-2 is an anti-inflammatory cytokine.

4) Introduction: Give a bit more detail about how CsA prevents activation of T cells via cyclophilins.

5) Introduction: The authors mention that CsA induced nephrotoxicity has been documented in both humans and animals, and then go on to describe a few of these studies. Please distinguish which studies were performed in humans, and which in animals. Is this an exhaustive reporting on known literature?

6) Introduction: It is known that NADPH oxidase (Nox) has a function in normal physiology. Elaborate why excessive levels of Nox are bad. Has this been documented in humans? What is the etiology of excessive Nox and the related pathophysiology in a diseased state?

7) Introduction: Please elaborate more on the mechanism of action of apocynin.

8) Materials and methods: What is 'plethysmography'?

9) Materials and methods: The timeline of the animal study is a bit confusing. At what point was the study stopped, and the animals euthanized? Is it 14 days?

10) Materials and methods: Whats is Pulse wave velocity? Explain it's significance.

11) Materials and methods- Biochemical analysis of oxidative stress markers: Elaborate a bit more on the principle and technique of some of the assays used to measure these markers. T-AOC methods takes into account which specific antioxidants?

12) Materials and methods- Histopathological studies: Mention what kind of stain was used.

13) Results: All the titles in the results section should reflect actual experimental findings instead of generic titles such as 'Effect of...'. For example, use 'Apocynin and catalase treatment ameliorated reduction and body weight and water intake seen with CsA treated rats.'

14) Figures: It is highly recommended that the data presented in Table 1 and Table 2 be presented as graphs with histograms. Visual data has more of an impact than obscure numbers in a table. Table 1 can be a multi-paneled figure 1 and Table 2 can likewise be figure 2. The paper would thus have 5 figures instead of 3.

15) Figure 3: Make it Figure 5, and please include a colored image instead.

16) Figure 3: It would be good if the authors could perform staining of histopathological sections for Nox 4 and some other markers such as MDA, SOD etc. Again, visual data is more impactful.

14) Discussion: Line 354: ....treatment of CsA injury rats with apocynin and catalase for 14 days ameliorated renal function....; Please use 'dysfunction' instead of 'function'.

15) Discussion- Line 355: Check sentence construction-Please correct sentence and say that CsA induces.... a 'negative'impact on both renal function and structure.

16) Discussion: What is FeNa+?

17) Discussion: Please elaborate findings in all the studies that have been referenced. Some studies have been described, some not. These include findings from references 23, 24, 33, 34, 35, 44. Actually, give more details for all studies referenced in the discussion section. What experimental model were they conducted in?

18) Discussion: It seems that in ref 34 and 35, the effect of apocynin and catalase together has already been demonstrated. Is this true, or was it by single drug only?

19) Discussion: The authors have discussed their findings and compared them with studies done previously where either apocynin or catalase was used. It is confusing if there is a study where both have been combined previously? If not, then this is the novel aspect of their study, and the authors need to emphasize on this and highlight this properly in their discussion, as this is not immediately obvious.

20) Discussion: In the studies describing the effects of apocynin or catalase, which were done in humans? Were there any side effects observed?

21) Did the rats demonstrate any side effects besides the documented effects?

22) Conclusion: The statement in Line 448 where the authors say that their findings suggest that renal injury in CsA model was associated with hypertension.....This is not a novel finding, as the authors describe this in the introduction (Ref 4). They present it as a novel finding though.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Apr 16;15(4):e0231472. doi: 10.1371/journal.pone.0231472.r002

Author response to Decision Letter 0

13 Dec 2019

Answers to reviewer’s comment

Paper No. PONE-D-19-26893

Title: “Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A induced nephrotoxicity in rats’’

PLOSONE

First of all, we would like to thank the Reviewers for their comments on our manuscript. We would also thank the editor for giving his consideration to this manuscript.

Reviewer #1:

The study evaluated the effect of apocynin and catalase in cyclosporine A-induced nephrotoxicity. Several biomarkers of oxidative stress were determined due to oxidative stress participated in nephrotoxicity as an important pathogenesis mechanism. The authors tested the MDA, SOD, NO and AOC level in plasma from different groups. Why do the authors test these biomarkers in the kidney tissue since the major tissue of injury induced by cyclosporine is the kidney tissue?

Authors’ response:

Thank you for the important suggestion of measuring MDA, SOD, NO and AOC levels in the kidney tissue. Measuring the levels of MDA, SOD, NO and AOC in the plasma helped us to study the contribution of biomarkers of oxidative stress in hypertension globally and it can be deduced that similar picture may exist locally within the kidney. In the present study, kidney tissue was used to observe the pathological changes in control and treatment groups and expression of NOX 4 mRNA. However, this concept of measuring the kidney levels of biomarkers of oxidative stress can be included in future studies along with their plasma levels to get more accurate and precise information both globally and locally.

Authors’ response:

As suggested by the Reviewer, measurement of blood urea nitrogen (BUN) was included in the manuscript at line numbers 148-151 in materials and method and lines 326-331 of results section of the current version of manuscript.

Authors’ response:

Thank you for the comment. The aim of the current study was to explore the expression of NOX4 mRNA in the kidney which ultimately predicts the expression of NOX4 protein. We agree that studying the NOX4 protein expression in the kidney tissue using IF or IHC would have an added value to the presentation in this manuscript and it is one of the limitations of this study. However, we believe that mRNA expression gives an equally important information about overexpression of this protein as a result of the disease process.

Reviewer#2

In the study, ‘Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats', the authors have demonstrated a potential therapeutic role for the agents combined, in treating harmful side effects associated with CsA treatment. While the study was designed and conducted well, there were some issues as highlighted below:

Authors’ response:

Author acknowledge and appreciate the Reviewer’s input regarding the abstract. In this version of the manuscript the abstract has been amended accordingly to include the required amendments.

2) Introduction: The authors say that CsA is a widely used immunosuppressant in organ transplantation. What is the rate of occurrence of nephrotoxicity in these patients clinically?

Authors’ response:

As suggested by the Reviewer, the incidence of cyclosporine induced nephrotoxicity has been incorporated in the current manuscript at lines 53-58.

3) Introduction: Mention that IL-2 is an anti-inflammatory cytokine.

Authors’ response:

Thank you. Interleukin-2 has now been mentioned as an inflammatory cytokine in line number 61-67.

4) Introduction: Give a bit more detail about how CsA prevents activation of T cells via cyclophilins.

Authors’ response:

As suggested by the Reviewer, effects of CsA in the activation of T cells via cyclophilins have now been mentioned in more details at lines 61-63.

Authors’ response:

Thank you. The present study emphasizes the effect of CsA induced nephrotoxicity in rats. Therefore, all the literature reported is mostly related to experimental animals. However, upon suggestions, we distinguished studies in rats from those in humans.

Authors’ response:

Studies in humans reported the effects of NOX4 on tissues such as coronory and other peripheral arteries contributing to atherosclerosis and endothelial dysfunction in these vessels respectively. However, the pattern of and physiological importance of NOX4 expression in human kidney is not fully elucidated. A clinical study depicted the presence of NOX5 in the renal vasculature (J Biol Chem. 2004, 279(18):18583-91) while another study showed correlation between NOX5 expression in the kidney and tissue injury in diabetic nephropathy (Diabetes. 2017, 66(10):2691-2703). The etiology of excessive NOX4 has been investigated before and it was found that it is related at least in part to transforming growth factor-beta (TGF-β) activity in a variety of cell types including endothelial cells and kidney cells (Circ Res. 2005, 97(9):900-7; Front Physiol. 2012; 3: 412). Similarly, it is also reported that tumor necrosis factor-alpha enhances the expression of NOX4 in a variety of vascular cells (Am J Physiol Cell Physiol. 2009, 296(3):C422-32). This has now been included in the introduction part as per the Reviewer’s comment.

7) Introduction: Please elaborate more on the mechanism of action of apocynin.

Authors’ response:

As suggested by the Reviewer, the mechanism of action of apocynin is added in the introduction of the current version of the manuscript lines 100-104.

8) Materials and methods: What is 'plethysmography'?

Authors’ response:

Blood pressure was measured non-invasively using the CODA plethysmograph system. CODA system uses a tail-cuff method to detect blood pressure based on volume changes in the tail. The term plethysmography refers to the method that uses changes in volume to detect changes in pressure. This has now been included under this section of the manuscript lines 154-159.

9) Materials and methods: The timeline of the animal study is a bit confusing. At what point was the study stopped, and the animals euthanized? Is it 14 days?

Authors’ response:

Study was terminated on day 14th and in order to avoid confusion, a clear statement is now included under Animals sub section of the materials and methods.

10) Materials and methods: What is pulse wave velocity? Explain its significance.

Authors’ response:

Pulse wave velocity is a widely used marker of arterial stiffness and was studied in many pathologies such as renal failure, hypertension, left ventricular hypertrophy and diabetes. In humans, pulse wave velocity is utilized as a good predictor of cardiovascular events and mortality. Pulse wave velocity significance is highlighted in the methods section at line number 185-189.

Authors’ response:

As suggested by the Reviewer, principle and detailed technique of all biochemical assays were incorporated at this point under respective sections of the manuscript as in line 217-221 for NO and line 225-227 for T-AOC.

12) Materials and methods- Histopathological studies: Mention what kind of stain was used.

Authors’ response:

Histopathology study was carried out using Hematoxyllin and Eosin staining and is now mentioned in respective part of the manuscript lines 232-235.

Authors’ response:

The authors fully agree with the Reviewer’s suggestion about the titles of the result sections. In this version of the manuscript we have amended the titles accordingly.

15) Figure 3: Make it Figure 5, and please include a colored image instead.

Authors’ response:

As suggested by the Reviewer, data in table 1 and 2 are now presented as Figures. Moreover, figure number is now amended accordingly, and colors were made consistent.

16) Figure 3: It would be good if the authors could perform staining of histopathological sections for Nox 4 and some other markers such as MDA, SOD etc. Again, visual data is more impactful.

Authors’ response:

Thank you for the comment. The aim of the current study was to explore the expression of NOX4 mRNA in the kidney which ultimately predicts the expression of NOX4 protein. We agree that histopathological studies of NOX4 and other markers in this study would have an added value to the presentation in this manuscript. However, we believe that mRNA expression gives an equally important information about overexpression of this protein as a result of the disease process. Moreover, the levels of oxidative stress markers in the plasma is still a reliable measure to reflect indirectly on the tissue expression of these markers. On the basis of these findings, this study demonstrated the effect of combined pharmacological intervention using apocynin and catalase on levels of these markers. The suggestion of the Reviewer will be very useful to broaden the scope of our future studies where we can conduct protein expression of the NOX, SOD, MDA mRNAs along with their IHC and IF assays which can be more informative to the readers. This study was conducted in best fashion while addressing these limitations.

14) Discussion: Line 354: ....treatment of CsA injury rats with apocynin and catalase for 14 days ameliorated renal function....; Please use 'dysfunction' instead of 'function'.

Authors’ response:

We apologize for this mistake from our side, it is now corrected.

15) Discussion- Line 355: Check sentence construction-Please correct sentence and say that CsA induces.... a 'negative'impact on both renal function and structure.

Authors’ response:

This sentence is now corrected as suggested by Reviewer. Thank you.

16) Discussion: What is FeNa+?

Authors’ response:

We apologize for the typo. In this version of the manuscript we have checked the fractional excretion of sodium abbreviation as FENa+ and amended accordingly as stated in line 144-145.

Authors’ response:

All references highlighted by the Reviewer are explained in the respective sections of the manuscript with the related model and findings. Thank you.

18) Discussion: It seems that in ref 34 and 35, the effect of apocynin and catalase together has already been demonstrated. Is this true, or was it by single drug only?

Authors’ response:

Thank you. Effects of apocynin and catalase were explored in models of kidney injury individually, but they were never been studied in combination in hypertension and kidney injury due to CsA. Moreover, exploration of the NOX4 mRNA in this model after treatment with either apocynin, catalase or a combination of both have never been studied which laid the objective of the present study. A recent study by Ciarcia et al., 2015 (J Cell Biochem. 2015, 116(9):1848-56) showed a protective effect of only apocynin on CsA renal injury without looking at NOX4 mRNA expression and without the use of catalase. Therefore, the present study presents a novel finding that a combined treatment approach is more potent than individual treatment and looked at potential mechanisms for this added effect through NOX4 mRNA expression in the kidney.

Authors’ response:

As stated above, we are unaware of a study which showed the combined effect of apocynin and catalase on NOX4 expression and on renal function and haemodynamics in CsA model of rats. Discussion is now modified where the novelty of this study was emphasized as per the Reviewer’s suggestion.

20) Discussion: In the studies describing the effects of apocynin or catalase, which were done in humans? Were there any side effects observed?

Authors’ response:

Thank you. The authors are unaware of any studies where the role of either apocynin or catalase was examined in humans as these agents are not approved from FDA for human use. However, numerous studies have been conducted on animals to collect scientific information related to both agents. Animals in the present study did not show any sign of toxicity with the dosage level used.

21) Did the rats demonstrate any side effects besides the documented effects?

Authors’ response:

Rats in the present study were monitored daily and did not show any adverse effects. The doses utilized in the present study were also used in previous studies with no reported side effects.

Authors’ response:

The authors fully agree with the Reviewer regarding novelty of hypertension in CsA model. The conclusion of the study is now adjusted to highlight novelty of the present study specifically with regards to combined treatment with apocynin and catalase.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(29.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0231472.r003

Decision Letter 1

Partha Mukhopadhyay

23 Jan 2020

PONE-D-19-26893R1

Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats

PLOS ONE

Dear Mr TAN SAMUAL,

Your manuscript was reviewed by same reviewers and one reviewer raised valid points , which require attention.during revision. Oxidative stress marker(s) in kidney tissue are essential to support the hypothesis of the manuscript.

We would appreciate receiving your revised manuscript by Mar 08 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Partha Mukhopadhyay, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: NADPH oxidases are a major source of reactive oxygen species (ROS) in the kidney in normal and pathological conditions. Among NADPH oxidase isoforms, NADPH oxidase4 (Nox4) is highly expressed in the kidney and has an important role in kidney diseases.

Nox4 is a NAPDH oxidase expressed in the kidney and has an important role in kidney function injury. In the study, the authors only detected the mRNA expression of Nox4 in kidney tissues from control and treatment groups. mRNA expression didn’t completely reflect the protein level and the activity of the protein. How the authors can conclude that the renal injury induced by CsA was associated with the increase in NADPH oxidase activity? In addition, the authors only detect the oxidative stress makers in plasma not the kidney tissue, how to confirm that apocynin and catalase prevent the rental injury through regulating the Nox4 and oxidative stress?

Reviewer #2: I recommend this manuscript for publication, however, I recommend a couple of minor edits:

1) Abstract: Add these words in the last sentence: 'NADPH inhibition and H202 scavenging could, therefore, be a potentially important therapeutic strategy to treat CsA-induced nephrotoxicity and hypertension.'

2) Abstract: Expand on human studies in references 6-8.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2020 Apr 16;15(4):e0231472. doi: 10.1371/journal.pone.0231472.r004

Author response to Decision Letter 1

7 Mar 2020

Answers to reviewer’s comment

Paper No. PONE-D-19-26893

Title: “Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A induced nephrotoxicity in rats’’

PLOSONE

First of all, we would like to thank the Reviewers for their comments on our manuscript. We also thank the Editor for giving his consideration to this manuscript.

Reviewer #1:

NADPH oxidases are a major source of reactive oxygen species (ROS) in the kidney in normal and pathological conditions. Among NADPH oxidase isoforms, NADPH oxidase4 (Nox4) is highly expressed in the kidney and has an important role in kidney diseases.

Nox4 is a NAPDH oxidase expressed in the kidney and has an important role in kidney function injury. In the study, the authors only detected the mRNA expression of Nox4 in kidney tissues from control and treatment groups. mRNA expression didn’t completely reflect the protein level and the activity of the protein. How the authors can conclude that the renal injury induced by CsA was associated with the increase in NADPH oxidase activity? In addition, the authors only detect the oxidative stress makers in plasma not the kidney tissue, how to confirm that apocynin and catalase prevent the renal injury through regulating the Nox4 and oxidative stress?

Authors’ response:

The aim of the study was to explore the status of expression of NADPH oxidase in cyclosporine model of kidney injury. The study emphasized that inhibition of this enzyme by administration of apocyanin and catalase will not only downregulate the expression of this enzyme but also will minimize the oxidative stress in the kidney. We showed that NADPH oxidase was indeed downregulated in the kidney by measuring NADPH oxidase mRNA by using qPCR. This downregulation of NADPH oxidase is suggested to translate into decreased protein levels of NADPH oxidase then reduction of oxidative stress biomarkers at the cellular level and in the plasma. As suggested by the Reviewer, we measured the oxidative stress biomarkers in the kidney tissue to relate the expression of NADPH oxidase mRNA with biomarker of oxidative stress. The results showed a similar trend in terms of tissue oxidative stress level as it was shown in the plasma. The levels of oxidative stress biomarkers in different groups are shown in updated Figures 4A, 4B and 4C in the updated version of the manuscript. The present study however presents the limitation that it measured NADPH oxidase mRNA and not the activity of this protein but we believe it highlighted that mRNA expression is altered in this renal injury model and in association with hypertension. Indeed, this study provides the bases for future studies to explore this gap in knowledge.

Reviewer #2: I recommend this manuscript for publication, however, I recommend a couple of minor edits:

Authors’ response:

Thank you indeed for the recommending this manuscript for publication. Suggested changes regarding insertion of the above sentence has now been done.

2) Introduction: Expand on human studies in references 6-8.

Authors’ response:

Suggested changes have been done in the introduction section of this manuscript.

Attachment

Submitted filename: 2nd Rebuttal.docx

Click here for additional data file.^{(14.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0231472.r005

Decision Letter 2

Partha Mukhopadhyay

25 Mar 2020

Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats

PONE-D-19-26893R2

Dear Dr. TAN SAMUAL,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Partha Mukhopadhyay, Ph.D.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors presented 23 figures in the manuscript. I think the authors should arrange these figures in Figure 1- Figure 5. In every figure, the authors should arrange the panel at A, B, C, D, E to present these results.

For example, In the results titled with “Apocynin and catalase restored renal excretory function in CsA rats’’, because all these figures presented the effect of Apo and Cat on rental function, Fig 3 to Fig 9 can be arranged at the same panel at A, B, C, D, E, F, G.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. doi: 10.1371/journal.pone.0231472.r006

Acceptance letter

Partha Mukhopadhyay

27 Mar 2020

PONE-D-19-26893R2

Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats

Dear Dr. TAN SAMUAL:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Partha Mukhopadhyay

Section Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(29.5KB, docx)}

Attachment

Submitted filename: 2nd Rebuttal.docx

Click here for additional data file.^{(14.5KB, docx)}

Data Availability Statement

All relevant data are within the paper.

[pone.0231472.ref001] 1.Yoon HE, Yang CW. Established and newly proposed mechanisms of chronic cyclosporine nephropathy. The Korean journal of internal medicine. 2009;24(2):81 10.3904/kjim.2009.24.2.81 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref002] 2.Ojo AO, Held PJ, Port FK, Wolfe RA, Leichtman AB, Young EW, et al. Chronic renal failure after transplantation of a nonrenal organ. New England Journal of Medicine. 2003;349(10):931–40. 10.1056/NEJMoa021744 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref003] 3.Colgan J, Asmal M, Yu B, Luban J. Cyclophilin A-deficient mice are resistant to immunosuppression by cyclosporine. The Journal of Immunology. 2005;174(10):6030–8. 10.4049/jimmunol.174.10.6030 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref004] 4.Friedman J, Weissman I. Two cytoplasmic candidates for immunophilin action are revealed by affinity for a new cyclophilin: one in the presence and one in the absence of CsA. Cell. 1991;66(4):799–6. 10.1016/0092-8674(91)90123-g [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref005] 5.Bobadilla NA, Gamba G. New insights into the pathophysiology of cyclosporine nephrotoxicity: a role of aldosterone. American Journal of Physiology-Renal Physiology. 2007;293(1):F2–F9. 10.1152/ajprenal.00072.2007 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref006] 6.Heering P, Schadewaldt P, Bach D, Grabensee B. Nephrotoxicity of cyclosporine in humans: effect of cyclosporine on glomerular filtration and proximal tubular reabsorption. The clinical investigator. 1993;71(12):1010–5. 10.1007/bf00180033 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref007] 7.Smith SR, Creech EA, Schaffer AV, Martin LL, Rakhit A, Douglas FL, et al. Effects of thromboxane synthase inhibition with CGS 13080 in human cyclosporine nephrotoxicity. Kidney international. 1992;41(1):199–5. 10.1038/ki.1992.27 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref008] 8.Burdmann EA, Andoh TF, Yu L, Bennett WM. Cyclosporine nephrotoxicity. Seminars in nephrology. 2003;23:465–76. 10.1016/s0270-9295(03)00090-1 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref009] 9.Chia TY, Sattar MA, Abdulla MH, Rathore HA, Ahmad FuD, Kaur G, et al. The effects of tempol on renal function and hemodynamics in cyclosporine-induced renal insufficiency rats. Renal failure. 2013;35(7):978–88. 10.3109/0886022X.2013.809563 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref010] 10.Ciresi DL, Lloyd MA, Sandberg SM, Heublein DM, Edwards BS. The sodium retaining effects of cyclosporine. Kidney international. 1992;41(6):1599–605. 10.1038/ki.1992.231 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref011] 11.Pichler RH, Franceschini N, Young BA, Hugo C, Andoh TF, Burdmann EA, et al. Pathogenesis of cyclosporine nephropathy: roles of angiotensin II and osteopontin. Journal of the American Society of Nephrology. 1995;6(4):1186–96. [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref012] 12.Modlinger P, Chabrashvili T, Gill PS, Mendonca M, Harrison DG, Griendling KK, et al. RNA silencing in vivo reveals role of p22phox in rat angiotensin slow pressor response. Hypertension. 2006;47(2):238–44. 10.1161/01.HYP.0000200023.02195.73 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref013] 13.De Keulenaer WG, Alexander RW, Ushio-Fukai M, Ishizaka N, Griendling KK. Tumour necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle. Biochemical journal. 1998;329(3):653–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref014] 14.Brandes RP, Viedt C, Nguyen K, Beer S, Kreuzer J, Busse R, et al. Thrombin-induced MCP-1 expression involves activation of the p22phox-containing NADPH oxidase in human vascular smooth muscle cells. Thrombosis and haemostasis. 2001;85(06):1104–10. [PubMed] [Google Scholar]

[pone.0231472.ref015] 15.Lassegue B, Clempus RE. Vascular NAD (P) H oxidases: specific features, expression, and regulation. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 2003;285(2):R277–R97. 10.1152/ajpregu.00758.2002 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref016] 16.Guzik TJ, Chen W, Gongora MC, Guzik B, Lob HE, Mangalat D, et al. Calcium-dependent NOX5 nicotinamide adenine dinucleotide phosphate oxidase contributes to vascular oxidative stress in human coronary artery disease. Journal of the American College of Cardiology. 2008;52(22):1803–9. 10.1016/j.jacc.2008.07.063 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref017] 17.Guzik TJ, Sadowski J, Guzik B, Jopek A, Kapelak B, Przybylowski P, et al. Coronary artery superoxide production and nox isoform expression in human coronary artery disease. Arteriosclerosis, thrombosis, and vascular biology. 2006;26(2):333–9. 10.1161/01.ATV.0000196651.64776.51 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref018] 18.Banfi B, Tirone F, Durussel I, Knisz J, Moskwa P, Molnar GZ, et al. Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). Journal of Biological Chemistry. 2004;279(18):18583–91. 10.1074/jbc.M310268200 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref019] 19.Jha JC, Banal C, Okabe J, Gray SP, Hettige T, Chow BSM, et al. NADPH oxidase Nox5 accelerates renal injury in diabetic nephropathy. Diabetes. 2017;66(10):2691–703. 10.2337/db16-1585 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref020] 20.Cucoranu I, Clempus R, Dikalova A, Phelan PJ, Ariyan S, Dikalov S, et al. NAD (P) H oxidase 4 mediates transforming growth factor-Beta1-induced differentiation of cardiac fibroblasts into myofibroblasts. Circulation research. 2005;97(9):900–7. 10.1161/01.RES.0000187457.24338.3D [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref021] 21.Chen F, Haigh S, Barman SA, Fulton D. From form to function: the role of Nox4 in the cardiovascular system. Frontiers in physiology. 2012;3:412 10.3389/fphys.2012.00412 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref022] 22.Parfenova H. Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF {alpha} in cerebral vascular endothelial cells Am J Physiol Cell Physiol 2009;296:C422–C32. 10.1152/ajpcell.00381.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref023] 23.Yang X, Zhao K, Deng W, Zhao L, Jin H, Mei F, et al. Apocynin Attenuates Acute Kidney Injury and Inflammation in Rats with Acute Hypertriglyceridemic Pancreatitis. Digestive Diseases and Sciences. 2019:1–13. [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref024] 24.Chia TY, Sattar MA, Abdullah MH, Ahmad FUD, Ibraheem ZO, Lia KJ, et al. Cyclosporine A—induced nephrotoxic Sprague-Dawley rats are more suscetable to altered vascular function and haemodynamic. International journal of pharmacy and pharmaceutical sciences. 2012;4:431–9. [Google Scholar]

[pone.0231472.ref025] 25.Ahmad FUD, Sattar MA, Rathore HA, Tan YC, Akhtar S, Jin OH, et al. Hydrogen sulphide and tempol treatments improve the blood pressure and renal excretory responses in spontaneously hypertensive rats. Renal failure. 2014;36(4):598–5. 10.3109/0886022X.2014.882218 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref026] 26.Ahmad FUD, Sattar MA, Rathore HA, Hussain AI, Chia TY, Jin OH, et al. Exogenous hydrogen sulfide attenuates oxidative stress in spontaneously hypertensive rats. International Journal of Pharmaceutical Sciences and Research. 2013;4(8):2916. [Google Scholar]

[pone.0231472.ref027] 27.Ahmad A, Sattar MA, Azam M, Khan SA, Bhatt O, Johns EJ. Interaction between nitric oxide and renal alpha1-adrenoreceptors mediated vasoconstriction in rats with left ventricular hypertrophyin Wistar Kyoto rats. PloS one. 2018;13(2):e0189386 10.1371/journal.pone.0189386 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref028] 28.Swarup KRLA, Sattar MA, Abdullah NA, Abdulla MH, Salman IM, Rathore HA, et al. Effect of dragon fruit extract on oxidative stress and aortic stiffness in streptozotocin-induced diabetes in rats. Pharmacognosy research. 2010;2(1):31–5. 10.4103/0974-8490.60582 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref029] 29.Vlachopoulos C, Aznaouridis K, Terentes-Printzios D, Ioakeimidis N, Stefanadis C. Prediction of cardiovascular events and all-cause mortality with brachial-ankle elasticity index: a systematic review and meta-analysis. Hypertension. 2012;60(2):556–62. 10.1161/HYPERTENSIONAHA.112.194779 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref030] 30.Wang Y-X, Halks-Miller M, Vergona R, Sullivan ME, Fitch R, Mallari C, et al. Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice. American Journal of Physiology-Heart and Circulatory Physiology. 2000;278(2):H428–H34. 10.1152/ajpheart.2000.278.2.H428 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref031] 31.Cosson E, Herisse M, Laude D, Thomas F, Valensi PE, Attali J-R, et al. Aortic stiffness and pulse pressure amplification in Wistar-Kyoto and spontaneously hypertensive rats. American Journal of Physiology-Heart and Circulatory Physiology. 2007; 292(5):H2506–12 10.1152/ajpheart.00732.2006 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref032] 32.Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical biochemistry. 1979;95(2):351–8. 10.1016/0003-2697(79)90738-3 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref033] 33.Oyanagui Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. Analytical biochemistry. 1984;142(2):290–6. 10.1016/0003-2697(84)90467-6 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref034] 34.Archer S. Measurement of nitric oxide in biological models. The FASEB journal. 1993;7(2):349–60. 10.1096/fasebj.7.2.8440411 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref035] 35.Sun J, Zhang X, Broderick M, Fein H. Measurement of nitric oxide production in biological systems by using Griess reaction assay. Sensors. 2003;3(8):276–84. [Google Scholar]

[pone.0231472.ref036] 36.Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clinical science. 1993;84(4):407–12. 10.1042/cs0840407 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref037] 37.Kitiyakara C, Chabrashvili T, Chen Y, Blau J, Karber A, Aslam S, et al. Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase. Journal of the American Society of Nephrology. 2003;14(11):2775–82. 10.1097/01.asn.0000092145.90389.65 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref038] 38.Ahmad A, Sattar MA, Rathore HA, Abdulla MH, Khan SA, Azam M, et al. Up Regulation of cystathione y lyase and Hydrogen Sulphide in the Myocardium Inhibits the Progression of Isoproterenol-Caffeine Induced Left Ventricular Hypertrophy in Wistar Kyoto Rats. PloS one. 2016;11(3):e0150137 10.1371/journal.pone.0150137 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref039] 39.Morphake P, Bariety J, Darlametsos I, Tsipas G, Gkikas G, Hornysh A, et al. Alteration of cyclosporine (CsA)-induced nephrotoxicity by gamma linolenic acid (GLA) and eicosapentaenoic acid (EPA) in wistar rats. Prostaglandins, leukotrienes and essential fatty acids. 1994;50(1):29–35. [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref040] 40.Chia TY, Sattar MA, Rathore HA, Abdullah MH, Singh GKC, Ahmad FUD, et al. The effects of tempol on renal function and hemodynamics in cyclosporine-induced renal insufficiency rats. Renal failure. 2013;35(7):978–88. 10.3109/0886022X.2013.809563 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref041] 41.Fassi A, Sangalli F, Colombi F, Perico N, Remuzzi G, Remuzzi A. Beneficial effects of calcium channel blockade on acute glomerular hemodynamic changes induced by cyclosporine. American journal of kidney diseases. 1999;33(2):267–75. 10.1016/s0272-6386(99)70299-4 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref042] 42.Hagar HH, El Etter E, Arafa M. Taurine attenuates hypertension and renal dysfunction induced by cyclosporine A in rats. Clinical and experimental pharmacology and physiology. 2006;33(3):189–96. 10.1111/j.1440-1681.2006.04345.x [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref043] 43.Naesens M, Kuypers DRJ, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clinical Journal of the American Society of Nephrology. 2009;4(2):481–8. 10.2215/CJN.04800908 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref044] 44.Campese VM, Kogosov E. Renal afferent denervation prevents hypertension in rats with chronic renal failure. Hypertension. 1995;25(4):878–82. [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref045] 45.Hausberg M, Kosch M, Harmelink P, Barenbrock M, Hohage H, Kisters K, et al. Sympathetic nerve activity in end-stage renal disease. Circulation. 2002;106(15):1974–9. 10.1161/01.cir.0000034043.16664.96 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref046] 46.Ciarcia R, Damiano S, Florio A, Spagnuolo M, Zacchia E, Squillacioti C, et al. The protective effect of apocynin on cyclosporine A-induced hypertension and nephrotoxicity in rats. Journal of cellular biochemistry. 2015;116(9):1848–56. 10.1002/jcb.25140 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref047] 47.Sousa T, Oliveira S, Afonso J, Morato M, Patinha D, Fraga S, et al. Role of H₂O₂ in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II. British journal of pharmacology. 2012;166(8):2386–401. 10.1111/j.1476-5381.2012.01957.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref048] 48.Hoorn EJ, Walsh SB, McCormick JA, Zietse R, Unwin RJ, Ellison DH. Pathogenesis of calcineurin inhibitor-induced hypertension. Journal of Nephrology. 2012;25(3):269–75. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref049] 49.Edemir B, Reuter S, Borgulya R, Schroter R, Neugebauer U, Gabriels G, et al. Acute rejection modulates gene expression in the collecting duct. Journal of the American Society of Nephrology. 2008;19(3):538–46. 10.1681/ASN.2007040513 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref050] 50.Kim SW, Wang W, Kwon T-H, Knepper MA, Frokiaer J, Nielsen S. Increased expression of ENaC subunits and increased apical targeting of AQP2 in the kidneys of spontaneously hypertensive rats. American Journal of Physiology-Renal Physiology. 2005; 289: F957–F68. 10.1152/ajprenal.00413.2004 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref051] 51.Ortiz PA, Garvin JL. Superoxide stimulates NaCl absorption by the thick ascending limb. American Journal of Physiology-Renal Physiology. 2002;283(5):F957–F62. 10.1152/ajprenal.00102.2002 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref052] 52.Juncos R, Garvin JL. Superoxide enhances Na-K-2Cl cotransporter activity in the thick ascending limb. American Journal of Physiology-Renal Physiology. 2005;288(5):F982–F7. 10.1152/ajprenal.00348.2004 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref053] 53.Bayorh MA, Rollins-Hairston A, Adiyiah J, Lyn D, Eatman D. Eplerenone inhibits aldosterone-induced renal expression of cyclooxygenase. Journal of the Renin-Angiotensin-Aldosterone System. 2012;13(3):353–9. 10.1177/1470320312443911 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref054] 54.Eudy RJ, Sahasrabudhe V, Sweeney K, Tugnait M, King-Ahmad A, Near K, et al. The use of plasma aldosterone and urinary sodium to potassium ratio as translatable quantitative biomarkers of mineralocorticoid receptor antagonism. Journal of translational medicine. 2011;9(1):180. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref055] 55.Zhang Z-H, Yu Y, Wei S-G, Felder RB. Centrally administered lipopolysaccharide elicits sympathetic excitation via NAD (P) H oxidase-dependent mitogen-activated protein kinase signaling. Journal of hypertension. 2010;28(4):806 10.1097/HJH.0b013e3283358b6e [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref056] 56.Altintas R, Polat A, Vardi N, Oguz F, Beytur A, Sagir M, et al. The protective effects of apocynin on kidney damage caused by renal ischemia/reperfusion. Journal of endourology. 2013;27(5):617–24. 10.1089/end.2012.0556 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref057] 57.Ma SF, Nishikawa M, Hyoudou K, Takahashi R, Ikemura M, Kobayashi Y, et al. Combining cisplatin with cationized catalase decreases nephrotoxicity while improving antitumor activity. Kidney international. 2007;72(12):1474–82. 10.1038/sj.ki.5002556 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref058] 58.Prevot A, Semama DS, Justrabo E, Guignard JP, Escousse A, Gouyon JB. Acute cyclosporine A-induced nephrotoxicity: a rabbit model. Pediatric nephrology. 2000;14(5):370–5. 10.1007/s004670050777 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref059] 59.Nishiyama A, Kobori H, Fukui T, Zhang G-X, Yao L, Rahman M, et al. Role of angiotensin II and reactive oxygen species in cyclosporine A-dependent hypertension. Hypertension. 2003;42(4):754–60. 10.1161/01.HYP.0000085195.38870.44 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref060] 60.Patel RS, Al Mheid I, Morris AA, Ahmed Y, Kavtaradze N, Ali S, et al. Oxidative stress is associated with impaired arterial elasticity. Atherosclerosis. 2011;218(1):90–5. 10.1016/j.atherosclerosis.2011.04.033 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref061] 61.Covic A, Mardare N, Gusbeth-Tatomir P, Buhaescu I, Goldsmith DJA. Acute effect of CyA A (Neoral (R)) on large artery hemodynamics in renal transplant patients. Kidney international. 2005;67(2):732–7. 10.1111/j.1523-1755.2005.67134.x [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref062] 62.Hamilton CA, Miller WH, Sammy A-B, Brosnan MJ, Drummond RD, McBride MW, et al. Strategies to reduce oxidative stress in cardiovascular disease. Clinical science. 2004;106(3):219–34. 10.1042/CS20030379 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref063] 63.Griendling KK. NADPH oxidases: new regulators of old functions. Antioxidants & redox signaling. 2006;8(9–10):1443–5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref064] 64.Griendling KK. Novel NAD (P) H oxidases in the cardiovascular system. Heart. 2004;90(5):491–3. 10.1136/hrt.2003.029397 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0231472.ref065] 65.Touyz RM. Apocynin, NADPH oxidase, and vascular cells: a complex matter. Hypertension. 2008; 51(2):172–4. 10.1161/HYPERTENSIONAHA.107.103200 [DOI] [PubMed] [Google Scholar]

[pone.0231472.ref066] 66.Taylor NE, Glocka P, Liang M, Cowley AW Jr. NADPH oxidase in the renal medulla causes oxidative stress and contributes to salt-sensitive hypertension in Dahl S rats. Hypertension. 2006;47(4):692–8. 10.1161/01.HYP.0000203161.02046.8d [DOI] [PubMed] [Google Scholar]

PERMALINK

Apocynin and catalase prevent hypertension and kidney injury in Cyclosporine A-induced nephrotoxicity in rats

Tan Yong Chia

Munavvar Abdul Sattar

Ahmad F Ahmeda

Nurzalina Abdul Karim Khan

Vikneswaran Murugaiyah

Ashfaq Ahmad

Zurina Hassan

Gurjeet Kaur

Mohammed Hadi Abdulla

Edward James Johns

Roles

Abstract

Introduction

Materials and methods

Animals

Metabolic and renal functional studies

Systolic blood pressure measurement

Hemodynamic study

General preparation and surgical procedure

Measurement of pulse wave velocity

Biochemical analysis for oxidative stress marker

Histopathological studies

Relative quantification of NOX4 mRNA expression in the kidney using StepOnePlus RT-PCR system

Results

Apocynin and catalase ameliorated reduction in body weight and water intake in CsA rats

Fig 1. Body weight values during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 2. Water intake during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Apocynin and catalase restored renal excretory function in CsA rats

Fig 3. Urine output during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 4. Creatinine clearance during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 5. Fractional excretion of sodium during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 6. Urinary Na+:K+ ratio during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 7. Urinary protein excretion during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 8. Kidney index at the end of 14 days study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 9. Blood urea nitrogen at the end of 14 days study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Apocynin and catalase treatment restored blood pressure and heart rate in CsA rats

Fig 10. Systolic blood pressure values during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 11. Heart rate values during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Apocynin and catalase restored renal and systemic hemodynamic parameters in CsA rats

Fig 13. Pulse wave velocity measured during acute experiment on Day 14 in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 14. β-Index measured during acute experiment on Day 14 in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Apocynin and catalase inhibited the increase in oxidative stress markers due to CsA

Fig 15. Plasma level of malondialdehyde (MDA) in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 16. Kidney level of malondialdehyde (MDA) in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 17. Plasma level of total superoxide dismutase activity (T-SOD) activity in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 18. Kidney level of superoxide dismutase activity (SOD) in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 19. Plasma level of nitric oxide (NO) activity in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 20. Plasma level of total antioxidant capacity (T-AOC) in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Fig 21. Kidney level of total antioxidant capacity (T-AOC) in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Apocynin and catalase restored renal Nox4 mRNA expression in CsA rats

Fig 22. The molecular expression of Nox 4 mRNAs in the renal cortex of control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.

Apocynin and catalase prevented structural alterations due to CsA-induced tissue damage

Discussion

Conclusion

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Partha Mukhopadhyay

Roles

Author response to Decision Letter 0

Decision Letter 1

Partha Mukhopadhyay

Roles

Author response to Decision Letter 1

Decision Letter 2

Partha Mukhopadhyay

Roles

Acceptance letter

Partha Mukhopadhyay

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Fig 6. Urinary Na⁺:K⁺ ratio during the study period in control (C), Apocynin (Apo), Catalase (Cat), Apocynin plus Catalase (ApoCat), CsA (Cx), CsA plus Apocynin (CxApo), CsA plus Catalase (CxCat), and CsA plus a combination of Apocynin and Catalase (CxApoCat) treated rats.