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Role of progesterone in melatonin-mediated protection against acute kidney injury Jyotsna Sehajpal,a Tajpreet Kaur,b Rajbir Bhatti,a and Amrit Pal Singha,* a b
Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab, India Department of Pharmacology, Khalsa College of Pharmacy, Amritsar, Punjab, India
article info
abstract
Article history:
Background: Melatonin is released by pineal gland and maintains circadian rhythm in the
Received 14 February 2014
body. It has been reported as renoprotective agent because of its antioxidant property.
Received in revised form
Recently, a cross talk between progesterone and melatonin has been observed in various
8 April 2014
preclinical studies. The present study investigated the involvement of progesterone re-
Accepted 9 April 2014
ceptors in melatonin-mediated protection against ischemia reperfusion induced acute
Available online xxx
kidney injury (AKI) in rats. Materials and methods: The rats were subjected to bilateral renal ischemia for 40 min fol-
Keywords:
lowed by reperfusion for 24 h to induce AKI. The AKI was assessed by measuring creatinine
Melatonin
clearance, serum urea, uric acid level, potassium level, fractional excretion of sodium,
Progesterone
lactate dehydrogenase activity, and microproteinuria. The oxidative stress in renal tissues
Kidney
was assessed by quantification of myeloperoxidase activity, thiobarbituric acid reactive
Ischemia
substances, superoxide anion generation, reduced glutathione level, and catalase activity.
Oxidative stress
The hematoxylineeosin staining was carried out to observe histopathologic changes in renal tissues. The melatonin (4 and 10 mg/kg, intraperitoneally) and progesterone receptor antagonist mifepristone (5 mg/kg, intraperitoneally) were used in the present study. Results: The renal ischemia reperfusion induced AKI as indicated by significant change in serum, urinary, and tissue parameters that was ameliorated by prior treatment with melatonin. No significant difference in serum progesterone level was observed between various groups used in the present study. The prior administration of mifepristone abolished melatonin-mediated protection against AKI. Conclusions: It is concluded that melatonin treatment affords protection against ischemia reperfusion induced AKI. Moreover, progesterone receptors are essentially involved in mediating protective role of melatonin against AKI in rats. ª 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Acute kidney injury (AKI) is characterized by reduced glomerular filtration rate and sudden decline in kidney function that results in accumulation of nitrogenous and other biochemical wastes in body. AKI accounts for 1% of hospital admissions and around 7% in hospitalized patients [1]. The patients with AKI
have mortality rate of 10% that increases to 80% in cases with renal replacement therapy. AKI is a major risk factor for nonrenal complications such as sepsis, delirium, and respiratory failure [2]. Ischemia is a state of blood and oxygen deprivation to a tissue resulting in reduced washout of metabolites. The reperfusion resumes blood supply to ischemic areas but itself initiates cascade of adverse reactions that cause damage to the
* Corresponding author. Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143005, Punjab, India. Tel.: þ91183-2258802-09x3467, þ91 985 545 5354; fax: þ91 183225 8819. E-mail address: [email protected] (A.P. Singh). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.04.025
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tissue. The ischemiaereperfusion is well documented to produce damage of various organs such as brain, heart, skeletal muscles, and kidneys. The ischemiaereperfusion is one of the prominent reasons for renal damage, and it is observed in various clinical conditions such as severe hypotension and subsequent resuscitation, kidney transplantation, and aortic vascular surgeries. The ischemiaereperfusion accounts for 30% of total cases of delayed graft dysfunction after renal transplantation [3]. The ischemiaereperfusion in kidneys results in depletion of adenosine triphosphate that directly causes disruption of cytoskeleton and alteration in polarity of cells thereby leading to their death. Moreover, it activates other cellular systems like endothelial cells and leukocytes that lead to renal damage [4]. N-acetyl-5-methoxytryptamine or melatonin is mainly synthesized by pineal gland. Apart from this, other organs such as eyes [5], brain [6], gut [7], bone marrow [8], skin [9] and immune cells [10] are also involved in production of melatonin. The beneficial roles of melatonin include correction of disrupted circadian rhythm, improvement of sleep disorders, regulation of immunomodulatory cytokines, anticancer activity, protective effect in cardiovascular complications, modulation of neuronal activity with anticonvulsant activity, and improvement in migraine therapy [11]. The melatonin has been reported to be protective against AKI induced by various procedures including ischemiaereperfusion [12], ureteral obstruction [13], renovascular hypertension [14], gentamicin [15], acetaminophen [16], and cyclosporine-induced nephropathy [17], which is attributed to its antioxidant activity. Progesterone plays a major role in synthesis of androgens and regulates mammalian reproduction. Apart from its role in reproductive physiology, the progesterone administration is reported to protect against focal cerebral IRI and neurologic defects. It is reported to reduce membrane lipid peroxidation against traumatic brain injury and possesses significant antioxidant activity [18]. Apart from brain, progesterone is well documented to protect against injuries of various organs including heart [19]. A recent study from our laboratory observed protective role of exogenously administered progesterone in ischemia reperfusion induced AKI in rats [20]. The cross talk between melatonin and progesterone is well documented [21,22]. The melatonin is reported to increase progesterone level and expression of progesterone receptors in reproductive tissues [21,23,24]. However, the impact of this cross talk on nonreproductive organs such as kidney has never been explored so far. The present study has been designed to investigate the possible involvement of progesterone receptors in melatonin-mediated protection against ischemia reperfusion induced AKI in rats.
allowed to acclimatize in metabolic cages for 24 h before subjecting them to surgical treatment. The AKI was induced by bilateral ischemiaereperfusion model in rats. The rats were anaesthetized with ketamine (50 mg/kg, intraperitoneally [i.p.]) and xylazine (10 mg/kg, i.p.). The anaesthetized rats were placed on surgical platform in dorsal position and rectal temperature was maintained at 37 C throughout the experimental procedure. Both kidneys were exposed through flank incisions, and renal pedicles were occluded using bulldog clamp for 40 min. The clamps were then removed to start reperfusion for next 24 h. The surgical site was sealed by continuous sutures in two layers. In sham group, the animals were exposed to similar surgical procedure except for occlusion of renal pedicles. The animals were returned to their metabolic cages for urine collection. After 24 h, the rats were anaesthetized using ketamine (50 mg/kg, i.p.). The blood samples were collected using retroorbital puncture, and rats were sacrificed by cervical dislocation. The serum isolated from blood was used for estimation of creatinine, urea, uric acid, sodium, potassium level, and lactate dehydrogenase (LDH) activity. Moreover, the creatinine, sodium, and protein content in urine were estimated. The kidneys were removed and washed with saline. A part of renal tissue was preserved for histopathologic studies, the small portion was used for estimation of superoxide anion generation (SAG), and the rest of tissue was minced and homogenized (10% wt/vol) in 1.17% potassium chloride solution using teflon homogenizer. The contents were centrifuged at 800 g for 20 min. The pellet obtained was used for the estimation of myeloperoxidase (MPO) activity, whereas the clear supernatant was used to estimate lipid peroxides in terms of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) level, and catalase (CAT) activity.
2.
2.3. level
Materials and methods
The present study was carried out in accordance with the guidelines framed by committee for the purpose of control and supervision of experiments on animals, Ministry of Environment and Forests, Government of India. Male Wistar rats weighing 200e250 g were used in the present study. They were maintained on standard chow and water ad libitum and were exposed to 12-h light and dark cycle. The rats were
2.1.
Estimation of serum progesterone level
The serum progesterone level was estimated by liquid phase radioimmunoassay using established procedure [20].
2.2.
Estimation of creatinine clearance
The serum and urine creatinine levels were assayed by alkaline picrate method using creatinine assay kit (Span Diagnostics Ltd, Surat, India). The creatinine clearance (CrCl) was calculated using the formula: [CrCl ¼ urine creatinine urine flow rate/plasma creatinine]. The results were expressed as milliliter per minute per kilogram of rat weight.
Estimation of serum urea, uric acid, and potassium
The urea, uric acid, and potassium levels were assayed using Q3 commercially available kit (Span Diagnostics Ltd,and Crest Biosystems, India). The values of urea and uric acid were expressed as milligram per deciliter of serum, whereas serum potassium level was expressed as millimoles per liter of serum.
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2.4.
Estimation of fractional excretion of sodium
The serum and urine sodium levels were estimated using commercially available kit (Crest Biosystems). The fractional excretion of sodium (FeNa) was calculated using formula: [FeNa ¼ (urine sodium/plasma sodium) (plasma creatinine/urine creatinine) 100]. The results were expressed as percentage change in values.
2.5.
Estimation of LDH activity
The estimation of LDH activity in serum samples was done by commercially available kit by Crest Biosystems. The LDH activity was expressed in unit per liter of serum.
2.6.
Estimation of microproteinuria
The protein content in urine was estimated using commercially available kit based on pyrogallol red method (Crest Biosystems). The results were expressed as milligrams per day.
2.7.
Estimation of oxidative stress in renal tissues
The oxidative stress in renal tissues was assessed by measuring MPO activity, TBARS, SAG, GSH level, and CAT activity. The MPO activity was measured using established procedure [25]. The MPO activity was expressed as unit per gram of tissue weight. The quantitative measurement of TBARS, an index of lipid peroxidation in kidney was performed [25]. The total protein content was estimated in the tissue homogenate using commercially available kit (Span Diagnostics Ltd). The results were expressed as nanomoles per milligram of protein. The SAG in renal tissue was assayed in terms of measuring reduced nitroblue tetrazolium (NBT) as previously described [26]. The results were expressed as reduced NBT picomoles per minute per milligram of tissue. The GSH content in renal tissue was estimated using method as previously described [26]. The results were expressed as micromoles of GSH per milligram of protein. The CAT activity was estimated and expressed as micromoles of H2O2 oxidized per minute per milligram of protein [26].
2.8.
rats. Group 2 (sham operated): surgery was performed to expose both kidneys but ischemia was not given. Group 3 (ischemiaereperfusion injury, IRI): both kidneys were occluded for 40 min followed by reperfusion for 24 h. Group 4 (melatonin low dose): melatonin (4 mg/kg, i.p) was administered 30 min before subjecting rats to IRI. Group 5 (melatonin high dose): melatonin (10 mg/kg, i.p) was administered 30 min before subjecting rats to IRI. Group 6 (melatonin high dose þ mifepristone): mifepristone (5 mg/kg, i.p) was administered 1 h before subjecting rats to IRI followed by protocol as mentioned in group 5.
2.10.
Drugs and chemicals
Melatonin was purchased from SRL, India. Mifepristone Q4 was procured form Cipla Pharmaceuticals, Mumbai, India. Xylazine was purchased from Indian Immunologicals Ltd, Hyderabad, India. Ketamine was obtained from Neon Pharmaceuticals, Mumbai, India. Eosin, hematoxylin, and creatinine were purchased from S D Fine Chemicals Limited, Mumbai, India. GSH and NBT were obtained from Loba Chemie, Mumbai, India. All other reagents used in the study were of analytical grade.
2.11.
Statistical analysis
Results were expressed as mean standard error of the mean. The data obtained from various groups were statistically Q5 analyzed using one-way analysis of variance followed by Tukey Kramer post hoc test using Graphpad InStat software. The P < 0.05 was considered to be statistically significant.
3.
Results
No significant difference between control and sham group was observed in various parameters used in the present study. Therefore, the data obtained in control group were used for further statistical analysis.
Hematoxylin and eosin staining
The renal tissues preserved in 10% neutral buffered formalin were dehydrated in graded concentrations of ethanol, immersed in xylene, and then embedded in paraffin. The sections of 4 mm thickness were cut and stained with hematoxylin and eosin. The slides were observed for gross histopathologic changes including increase in tubulointerstitial space, tubular dilatation, cellular necrosis, and detachment of basement membrane from glomerulus.
3.1. Effect of melatonin and mifepristone on serum progesterone level No significant difference in serum progesterone level was observed between various groups used in the present study (Table 1).
3.2. 2.9.
3
Effect of melatonin and mifepristone on CrCl
Experimental protocol
Six groups were used in the present study, each containing six rats. Melatonin was dissolved in minimal volume of ethanol and final volume was made with normal saline. Mifepristone was dissolved in normal saline and administered to rats. Group 1 (control): no surgery was performed on
A significant decrease in CrCl was observed in IRI group compared with control group. The melatonin treatment significantly ameliorated IRI-induced decrease in CrCl. The IRI þ melatonin þ mifepristone group demonstrated significant decrease in CrCl compared with IRI þ melatonin high dose group (Table 1).
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Table 1 e Effect of melatonin and mifepristone on renal parameters. Groups Parameters Serum progesterone (ng/mL) CrCl (mL/min/kg) Serum urea (mg/dL) Uric acid (mg/dL) Serum potassium (mM) FeNa (%) LDH activity (U/L) Microproteinuria (mg/day)
Control
1.21 0.13 1.32 16.11 0.39 1.45
0.13 0.85 0.08 0.24
1.27 0.23 262.41 7.78 3.51 0.83
Sham
IRI
IRI þ melatonin (4 mg/kg)
IRI þ melatonin (10 mg/kg)
IRI þ melatonin þ mifepristone
1.14 0.21
1.58 0.09a
1.42 0.15b
1.69 0.18b
1.75 0.30a,d
1.21 23.90 0.47 1.75
0.09 4.31 0.05 0.31
1.51 0.12 293.44 11.62 5.32 1.31
0.41 111.36 1.51 3.84
0.03a 10.32a 0.20a 0.40a
3.33 0.27a 453.97 21.12a 31.27 2.05a
0.81 43.21 0.794 2.17
0.06b 4.23b 0.08b 0.17b
2.08 0.13b 268.48 23.82b 18.61 1.03b
0.99 30.54 0.59 1.84
0.07b 4.87b 0.07b 0.23b
1.74 0.15b 277.25 19.79b 14.12 1.71b
0.52 86.03 1.36 3.38
0.04a,c,d 9.52a,c,d 0.11a,c,d 0.19a,c,d
2.95 0.19a,d 442.52 15.83a,c,d 29.92 1.29a,c,d
Values are expressed as mean standard error of the mean. a ¼ P < 0.05 versus control; b ¼ P < 0.05 versus IRI; c ¼ P < 0.05 versus IRI þ melatonin (4 mg/kg); d ¼ P < 0.05 versus IRI þ melatonin (10 mg/kg) group.
3.3. Effect of melatonin and mifepristone on serum urea, uric acid, and potassium level
3.7. Effect of melatonin and mifepristone on oxidative stress parameters
A significant increase in serum urea level was observed in IRI group compared with control group. The treatment with melatonin resulted in a significant decrease in urea level compared with IRI group. The IRI þ melatonin þ mifepristone group demonstrated significant increase in serum urea level compared with melatonin low as well as high dose treatment. Similar pattern of results was observed with serum uric acid and serum potassium level (Table 1).
A significant increase in MPO activity was observed in IRI group compared with control group. The melatonin treatment significantly decreased the MPO activity compared with IRI group. The IRI þ melatonin þ mifepristone group demonstrated significant increase in MPO activity compared with IRI þ melatonin low as well as high dose group (Table 2). A significant increase in lipid peroxidation measured in terms of TBARS was observed in IRI group compared with control group. The melatonin treatment significantly decreased the TBARS level compared with IRI group. There was a significant rise in TBARS level in IRI þ melatonin þ mifepristone group compared with IRI þ melatonin high dose group (Table 2). Similar pattern of results was observed in SAG measured in terms of reduced NBT in the present study. A significant reduction in renal GSH level was observed in IRI group compared with control group. The melatonin treatment significantly increased the GSH level compared with IRI group. The IRI þ melatonin þ mifepristone group revealed significant reduction in GSH level compared with IRI þ melatonin low as well as high dose group. Similar pattern of results was observed in CAT activity between various groups used in the present study (Table 2).
3.4.
Effect of melatonin and mifepristone on FeNa
A significant increase in FeNa was observed in IRI group compared with control group. The treatment with melatonin resulted in a significant decrease in FeNa compared with IRI group. The IRI þ melatonin þ mifepristone group witnessed significant increase in FeNa compared with IRI þ melatonin high dose group (Table 1).
3.5. Effect of melatonin and mifepristone on LDH activity A significant increase in LDH activity was observed in IRI group compared with control group. The melatonin treatment significantly decreased the LDH activity, whereas LDH activity increased significantly in IRI þ melatonin þ mifepristone group compared with IRI þ melatonin low as well as high dose group (Table 1).
3.6. Effect of melatonin and mifepristone on microproteinuria A significant increase in level of urinary proteins was observed in IRI group compared with control group. The melatonin treatment significantly decreased level of urinary proteins. The IRI þ melatonin þ mifepristone group demonstrated significant increase in microproteinuria compared with IRI þ melatonin (4 and 10 mg/kg) group (Table 1).
3.8. Histopathologic evaluation of the effect of melatonin and mifepristone on renal tissues The hematoxylin and eosin staining revealed detachment of glomeruli from basement membrane, increased interstitial spaces, cell debris in tubules and neutrophil accumulation in renal sections of IRI group compared with control group. The treatment with melatonin resulted in reversal of IRIinduced changes in renal tissues. However, the treatment with mifepristone abolished melatonin-mediated renoprotective effect (Fig).
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Table 2 e Effect of melatonin and mifepristone on renal oxidative stress parameters. Groups Parameters MPO (U/g of tissue) TBARS (nM/mg of protein) SAG (pM/min/mg of tissue) GSH (mM/mg of protein) CAT (mM of H2O2 oxidized/min/mg of protein)
Control
Sham
IRI
IRI þ melatonin (4 mg/kg)
IRI þ melatonin (10 mg/kg)
IRI þ melatonin þ mifepristone
0.50 0.10 0.12 0.01
0.52 0.08 0.15 0.01
1.62 0.22a 0.46 0.02a
0.70 0.15b 0.26 0.03b
0.57 0.12b 0.25 0.01b
1.47 0.26a,c,d 0.39 0.04a,d
27.31 1.32
28.43 2.54
43.71 3.67a
30.05 2.14b
25.33 1.57b
3.49 0.28 0.39 0.02
3.14 0.42 0.37 0.03
1.81 0.16a 0.06 0.01a
2.91 0.32b 0.35 0.02b
3.11 0.41b 0.37 0.05b
40.27 2.33a,d 1.71 0.23a,c,d 0.11 0.02a,c,d
Values are expressed as mean standard error of the mean. a ¼ P < 0.05 versus control; b ¼ P < 0.05 versus IRI; c ¼ P < 0.05 versus IRI þ melatonin (4 mg/kg); d ¼ P < 0.05 versus IRI þ melatonin (10 mg/kg) group.
4.
Discussion
Various pharmacologic strategies used for management of AKI include use of loop diuretics that prevent reabsorption of water and reduces volume overload, dopamine that increases renal blood flow by splanchnic vasodilatation thereby increasing cardiac output and reperfusion pressure, fenoldopam that acts by increasing renal blood flow, natriuretic peptides to maintain pressure and homeostasis by modulating cardiac and renal function, atrial natriuretic peptide associated with an increase in glomerular filtration and Nacetylcysteine that is an antioxidant with vasodilating properties [27e31]. The IRI involves temporary cessation of blood flow to an organ followed by its resumption. The oxidative stress is caused by generation of reactive oxygen species (ROS) during ischemiaereperfusion [32,33]. It is an autocatalytic process that involves oxidative destruction of cellular membrane and accumulation of inflammatory mediators that lead to cell death [34]. The renal ischemiaereperfusion is the leading
cause of AKI and is associated with a high rate of mortality despite of the advancements in therapy. Various surgical procedures such as renal transplantation, partial nephrectomy, and aortic aneurysm involve transient discontinuation of renal blood flow. The resumption of the blood flow may lead to IRI thus introducing significant postoperative renal complications. The IRI-induced AKI is characterized by tubular damage and cell necrosis associated with ROS and accumulation of inflammatory cells [35,36]. Melatonin is a hormone secreted by pineal gland and is mainly responsible for controlling circadian rhythm. Apart from this, the role of melatonin has been explored in large number of pathophysiological processes such as regulation of immunomodulatory cytokines, inhibition of apoptosis, oncostatic action, management of cardiovascular complications including atherosclerosis, hypertension, angina pectoris, and neurologic disorders such as epilepsy and migraine [11]. Clinically, a decrease in melatonin rhythm with advancement of renal dysfunction has been observed in patients with chronic kidney disease [37]. The melatonin treatment demonstrated protection against microproteinuria in diabetic patients [38].
Fig e Effect of melatonin and mifepristone on renal tissues observed with hematoxylineeosin staining at 3400 magnification. ([A] control; [B] IRI; [C] IRI D melatonin [4 mg/kg]; [D] IRI D melatonin [10 mg/kg]; [E] IRI D melatonin D mifepristone). The arrows denote increased interstitial spaces, “*” denotes cell debris in tubules, “#” denotes detachment of glomeruli from basement membrane, whereas “**” denotes inflammatory cells. (Color version of figure is available online). 5.2.0 DTD YJSRE12693_proof 30 April 2014 8:36 pm ce
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Further preclinical and clinical studies are warranted to explore molecular mechanisms of melatonin-mediated renoprotection and its benefits in patients with renal dysfunction, respectively. The present study explored the involvement of progesterone receptors in melatonin-mediated protection against renal IRI. The melatonin is well documented to protect against IRI of various organs including heart [39], brain [40], and liver [41]. The correction of serum, urinary, and tissue oxidative stress parameters observed with melatonin treatment in the present study is in accordance with earlier studies [12,42]. The reduction in MPO activity along with reduction in neutrophil accumulation observed in hematoxylineeosin staining indicated anti-inflammatory property of melatonin. Being an amphiphilic molecule, it is able to penetrate and scavenge the cells [42]. The replenishment of reduced glutathione and increased CAT activity with melatonin treatment is important as depleted endogenous defense mechanisms are reported to produce more damage than increased levels of free radicals [20]. The melatonin (10 mg/kg) administered through various routes is reported to be protective against renal IRI in various studies [43e45]. No significant difference between two doses of melatonin (4 and 10 mg/kg) was observed in the present study that indicates that 4 mg/kg is effective dose and it could be used in future studies rather than using 10 mg/kg. The protective effect of melatonin was abolished with prior administration of progesterone receptor antagonist. There are various reports indicating cross talk between progesterone and melatonin [21,22]. The progesterone is a hormone required for maintenance of reproductive functions such as ovulation and implantation in females and androgen biosynthesis in males. Apart from its role in reproductive physiology, the neuroprotective role of progesterone against cerebral IRI is reported [46,47]. The progesterone treatment is noted to reduce the level of excitatory amino acids. Moreover, it increases the level of inhibitory neurotransmitters such as gamma amino butyric acid in brain [48]. It is also noted to upregulate the number of gamma amino butyric acid receptors [49]. The progesterone is well documented to reduce ROS and inflammatory cytokines in various pathologic conditions. The presence of progesterone receptors on kidney indicates their role in regulation of renal function [50]. The study from our laboratory indicated protective role of progesterone against renal IRI in dose-dependent manner [20]. The melatonin is reported to increase the progesterone levels [22] and also the expression of progesterone receptors [21,24]. The present study did not observe significant increase in serum progesterone level with melatonin treatment in rats. The single administration of melatonin given to rats in present study may be insufficient to produce a significant increase in progesterone level as observed with other agents such as ascorbic acid [20]. Moreover, most of the studies reporting increase in progesterone level have been conducted in cell cultures where cells are exposed to high concentrations of melatonin [51]. Third, the studies are carried out in granulosa or follicular cells that are specialized cells to produce progesterone compared with male rats used in the present study [23,51,52]. However, the treatment with mifepristone totally abolished melatonin-mediated antioxidant and protection against renal IRI that clearly
indicates involvement of progesterone receptors in melatonin-mediated renoprotective effect. Hence, we conclude that melatonin exhibits antioxidant effect that accounts for its renoprotective effect. Moreover, the progesterone receptors are essentially involved in mediating protective role of melatonin against ischemia reperfusion induced AKI in rats.
Acknowledgment Authors’ contributions: A.P.S. conceived, designed, and supervised the project. J.S. performed pharmacologic treatments. J.S., T.K., and R.B. did biochemical and histologic studies. J.S. and A.P.S. analyzed the data and wrote the article. All authors approved the article before submission to journal.
Disclosure The authors do not have any conflict of interest.
references
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Role of progesterone in melatonin-mediated protection against acute kidney injury Jyotsna Sehajpal,a Tajpreet Kaur,b Rajbir Bhatti,a and Amrit Pal Singha,* a b
Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab, India Department of Pharmacology, Khalsa College of Pharmacy, Amritsar, Punjab, India
article info
abstract
Article history:
Background: Melatonin is released by pineal gland and maintains circadian rhythm in the
Received 14 February 2014
body. It has been reported as renoprotective agent because of its antioxidant property.
Received in revised form
Recently, a cross talk between progesterone and melatonin has been observed in various
8 April 2014
preclinical studies. The present study investigated the involvement of progesterone re-
Accepted 9 April 2014
ceptors in melatonin-mediated protection against ischemia reperfusion induced acute
Available online xxx
kidney injury (AKI) in rats. Materials and methods: The rats were subjected to bilateral renal ischemia for 40 min fol-
Keywords:
lowed by reperfusion for 24 h to induce AKI. The AKI was assessed by measuring creatinine
Melatonin
clearance, serum urea, uric acid level, potassium level, fractional excretion of sodium,
Progesterone
lactate dehydrogenase activity, and microproteinuria. The oxidative stress in renal tissues
Kidney
was assessed by quantification of myeloperoxidase activity, thiobarbituric acid reactive
Ischemia
substances, superoxide anion generation, reduced glutathione level, and catalase activity.
Oxidative stress
The hematoxylineeosin staining was carried out to observe histopathologic changes in renal tissues. The melatonin (4 and 10 mg/kg, intraperitoneally) and progesterone receptor antagonist mifepristone (5 mg/kg, intraperitoneally) were used in the present study. Results: The renal ischemia reperfusion induced AKI as indicated by significant change in serum, urinary, and tissue parameters that was ameliorated by prior treatment with melatonin. No significant difference in serum progesterone level was observed between various groups used in the present study. The prior administration of mifepristone abolished melatonin-mediated protection against AKI. Conclusions: It is concluded that melatonin treatment affords protection against ischemia reperfusion induced AKI. Moreover, progesterone receptors are essentially involved in mediating protective role of melatonin against AKI in rats. ª 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Acute kidney injury (AKI) is characterized by reduced glomerular filtration rate and sudden decline in kidney function that results in accumulation of nitrogenous and other biochemical wastes in body. AKI accounts for 1% of hospital admissions and around 7% in hospitalized patients [1]. The patients with AKI
have mortality rate of 10% that increases to 80% in cases with renal replacement therapy. AKI is a major risk factor for nonrenal complications such as sepsis, delirium, and respiratory failure [2]. Ischemia is a state of blood and oxygen deprivation to a tissue resulting in reduced washout of metabolites. The reperfusion resumes blood supply to ischemic areas but itself initiates cascade of adverse reactions that cause damage to the
* Corresponding author. Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143005, Punjab, India. Tel.: þ91183-2258802-09x3467, þ91 985 545 5354; fax: þ91 183225 8819. E-mail address: [email protected] (A.P. Singh). 0022-4804/$ e see front matter ª 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.04.025
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tissue. The ischemiaereperfusion is well documented to produce damage of various organs such as brain, heart, skeletal muscles, and kidneys. The ischemiaereperfusion is one of the prominent reasons for renal damage, and it is observed in various clinical conditions such as severe hypotension and subsequent resuscitation, kidney transplantation, and aortic vascular surgeries. The ischemiaereperfusion accounts for 30% of total cases of delayed graft dysfunction after renal transplantation [3]. The ischemiaereperfusion in kidneys results in depletion of adenosine triphosphate that directly causes disruption of cytoskeleton and alteration in polarity of cells thereby leading to their death. Moreover, it activates other cellular systems like endothelial cells and leukocytes that lead to renal damage [4]. N-acetyl-5-methoxytryptamine or melatonin is mainly synthesized by pineal gland. Apart from this, other organs such as eyes [5], brain [6], gut [7], bone marrow [8], skin [9] and immune cells [10] are also involved in production of melatonin. The beneficial roles of melatonin include correction of disrupted circadian rhythm, improvement of sleep disorders, regulation of immunomodulatory cytokines, anticancer activity, protective effect in cardiovascular complications, modulation of neuronal activity with anticonvulsant activity, and improvement in migraine therapy [11]. The melatonin has been reported to be protective against AKI induced by various procedures including ischemiaereperfusion [12], ureteral obstruction [13], renovascular hypertension [14], gentamicin [15], acetaminophen [16], and cyclosporine-induced nephropathy [17], which is attributed to its antioxidant activity. Progesterone plays a major role in synthesis of androgens and regulates mammalian reproduction. Apart from its role in reproductive physiology, the progesterone administration is reported to protect against focal cerebral IRI and neurologic defects. It is reported to reduce membrane lipid peroxidation against traumatic brain injury and possesses significant antioxidant activity [18]. Apart from brain, progesterone is well documented to protect against injuries of various organs including heart [19]. A recent study from our laboratory observed protective role of exogenously administered progesterone in ischemia reperfusion induced AKI in rats [20]. The cross talk between melatonin and progesterone is well documented [21,22]. The melatonin is reported to increase progesterone level and expression of progesterone receptors in reproductive tissues [21,23,24]. However, the impact of this cross talk on nonreproductive organs such as kidney has never been explored so far. The present study has been designed to investigate the possible involvement of progesterone receptors in melatonin-mediated protection against ischemia reperfusion induced AKI in rats.
allowed to acclimatize in metabolic cages for 24 h before subjecting them to surgical treatment. The AKI was induced by bilateral ischemiaereperfusion model in rats. The rats were anaesthetized with ketamine (50 mg/kg, intraperitoneally [i.p.]) and xylazine (10 mg/kg, i.p.). The anaesthetized rats were placed on surgical platform in dorsal position and rectal temperature was maintained at 37 C throughout the experimental procedure. Both kidneys were exposed through flank incisions, and renal pedicles were occluded using bulldog clamp for 40 min. The clamps were then removed to start reperfusion for next 24 h. The surgical site was sealed by continuous sutures in two layers. In sham group, the animals were exposed to similar surgical procedure except for occlusion of renal pedicles. The animals were returned to their metabolic cages for urine collection. After 24 h, the rats were anaesthetized using ketamine (50 mg/kg, i.p.). The blood samples were collected using retroorbital puncture, and rats were sacrificed by cervical dislocation. The serum isolated from blood was used for estimation of creatinine, urea, uric acid, sodium, potassium level, and lactate dehydrogenase (LDH) activity. Moreover, the creatinine, sodium, and protein content in urine were estimated. The kidneys were removed and washed with saline. A part of renal tissue was preserved for histopathologic studies, the small portion was used for estimation of superoxide anion generation (SAG), and the rest of tissue was minced and homogenized (10% wt/vol) in 1.17% potassium chloride solution using teflon homogenizer. The contents were centrifuged at 800 g for 20 min. The pellet obtained was used for the estimation of myeloperoxidase (MPO) activity, whereas the clear supernatant was used to estimate lipid peroxides in terms of thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) level, and catalase (CAT) activity.
2.
2.3. level
Materials and methods
The present study was carried out in accordance with the guidelines framed by committee for the purpose of control and supervision of experiments on animals, Ministry of Environment and Forests, Government of India. Male Wistar rats weighing 200e250 g were used in the present study. They were maintained on standard chow and water ad libitum and were exposed to 12-h light and dark cycle. The rats were
2.1.
Estimation of serum progesterone level
The serum progesterone level was estimated by liquid phase radioimmunoassay using established procedure [20].
2.2.
Estimation of creatinine clearance
The serum and urine creatinine levels were assayed by alkaline picrate method using creatinine assay kit (Span Diagnostics Ltd, Surat, India). The creatinine clearance (CrCl) was calculated using the formula: [CrCl ¼ urine creatinine urine flow rate/plasma creatinine]. The results were expressed as milliliter per minute per kilogram of rat weight.
Estimation of serum urea, uric acid, and potassium
The urea, uric acid, and potassium levels were assayed using Q3 commercially available kit (Span Diagnostics Ltd,and Crest Biosystems, India). The values of urea and uric acid were expressed as milligram per deciliter of serum, whereas serum potassium level was expressed as millimoles per liter of serum.
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2.4.
Estimation of fractional excretion of sodium
The serum and urine sodium levels were estimated using commercially available kit (Crest Biosystems). The fractional excretion of sodium (FeNa) was calculated using formula: [FeNa ¼ (urine sodium/plasma sodium) (plasma creatinine/urine creatinine) 100]. The results were expressed as percentage change in values.
2.5.
Estimation of LDH activity
The estimation of LDH activity in serum samples was done by commercially available kit by Crest Biosystems. The LDH activity was expressed in unit per liter of serum.
2.6.
Estimation of microproteinuria
The protein content in urine was estimated using commercially available kit based on pyrogallol red method (Crest Biosystems). The results were expressed as milligrams per day.
2.7.
Estimation of oxidative stress in renal tissues
The oxidative stress in renal tissues was assessed by measuring MPO activity, TBARS, SAG, GSH level, and CAT activity. The MPO activity was measured using established procedure [25]. The MPO activity was expressed as unit per gram of tissue weight. The quantitative measurement of TBARS, an index of lipid peroxidation in kidney was performed [25]. The total protein content was estimated in the tissue homogenate using commercially available kit (Span Diagnostics Ltd). The results were expressed as nanomoles per milligram of protein. The SAG in renal tissue was assayed in terms of measuring reduced nitroblue tetrazolium (NBT) as previously described [26]. The results were expressed as reduced NBT picomoles per minute per milligram of tissue. The GSH content in renal tissue was estimated using method as previously described [26]. The results were expressed as micromoles of GSH per milligram of protein. The CAT activity was estimated and expressed as micromoles of H2O2 oxidized per minute per milligram of protein [26].
2.8.
rats. Group 2 (sham operated): surgery was performed to expose both kidneys but ischemia was not given. Group 3 (ischemiaereperfusion injury, IRI): both kidneys were occluded for 40 min followed by reperfusion for 24 h. Group 4 (melatonin low dose): melatonin (4 mg/kg, i.p) was administered 30 min before subjecting rats to IRI. Group 5 (melatonin high dose): melatonin (10 mg/kg, i.p) was administered 30 min before subjecting rats to IRI. Group 6 (melatonin high dose þ mifepristone): mifepristone (5 mg/kg, i.p) was administered 1 h before subjecting rats to IRI followed by protocol as mentioned in group 5.
2.10.
Drugs and chemicals
Melatonin was purchased from SRL, India. Mifepristone Q4 was procured form Cipla Pharmaceuticals, Mumbai, India. Xylazine was purchased from Indian Immunologicals Ltd, Hyderabad, India. Ketamine was obtained from Neon Pharmaceuticals, Mumbai, India. Eosin, hematoxylin, and creatinine were purchased from S D Fine Chemicals Limited, Mumbai, India. GSH and NBT were obtained from Loba Chemie, Mumbai, India. All other reagents used in the study were of analytical grade.
2.11.
Statistical analysis
Results were expressed as mean standard error of the mean. The data obtained from various groups were statistically Q5 analyzed using one-way analysis of variance followed by Tukey Kramer post hoc test using Graphpad InStat software. The P < 0.05 was considered to be statistically significant.
3.
Results
No significant difference between control and sham group was observed in various parameters used in the present study. Therefore, the data obtained in control group were used for further statistical analysis.
Hematoxylin and eosin staining
The renal tissues preserved in 10% neutral buffered formalin were dehydrated in graded concentrations of ethanol, immersed in xylene, and then embedded in paraffin. The sections of 4 mm thickness were cut and stained with hematoxylin and eosin. The slides were observed for gross histopathologic changes including increase in tubulointerstitial space, tubular dilatation, cellular necrosis, and detachment of basement membrane from glomerulus.
3.1. Effect of melatonin and mifepristone on serum progesterone level No significant difference in serum progesterone level was observed between various groups used in the present study (Table 1).
3.2. 2.9.
3
Effect of melatonin and mifepristone on CrCl
Experimental protocol
Six groups were used in the present study, each containing six rats. Melatonin was dissolved in minimal volume of ethanol and final volume was made with normal saline. Mifepristone was dissolved in normal saline and administered to rats. Group 1 (control): no surgery was performed on
A significant decrease in CrCl was observed in IRI group compared with control group. The melatonin treatment significantly ameliorated IRI-induced decrease in CrCl. The IRI þ melatonin þ mifepristone group demonstrated significant decrease in CrCl compared with IRI þ melatonin high dose group (Table 1).
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Table 1 e Effect of melatonin and mifepristone on renal parameters. Groups Parameters Serum progesterone (ng/mL) CrCl (mL/min/kg) Serum urea (mg/dL) Uric acid (mg/dL) Serum potassium (mM) FeNa (%) LDH activity (U/L) Microproteinuria (mg/day)
Control
1.21 0.13 1.32 16.11 0.39 1.45
0.13 0.85 0.08 0.24
1.27 0.23 262.41 7.78 3.51 0.83
Sham
IRI
IRI þ melatonin (4 mg/kg)
IRI þ melatonin (10 mg/kg)
IRI þ melatonin þ mifepristone
1.14 0.21
1.58 0.09a
1.42 0.15b
1.69 0.18b
1.75 0.30a,d
1.21 23.90 0.47 1.75
0.09 4.31 0.05 0.31
1.51 0.12 293.44 11.62 5.32 1.31
0.41 111.36 1.51 3.84
0.03a 10.32a 0.20a 0.40a
3.33 0.27a 453.97 21.12a 31.27 2.05a
0.81 43.21 0.794 2.17
0.06b 4.23b 0.08b 0.17b
2.08 0.13b 268.48 23.82b 18.61 1.03b
0.99 30.54 0.59 1.84
0.07b 4.87b 0.07b 0.23b
1.74 0.15b 277.25 19.79b 14.12 1.71b
0.52 86.03 1.36 3.38
0.04a,c,d 9.52a,c,d 0.11a,c,d 0.19a,c,d
2.95 0.19a,d 442.52 15.83a,c,d 29.92 1.29a,c,d
Values are expressed as mean standard error of the mean. a ¼ P < 0.05 versus control; b ¼ P < 0.05 versus IRI; c ¼ P < 0.05 versus IRI þ melatonin (4 mg/kg); d ¼ P < 0.05 versus IRI þ melatonin (10 mg/kg) group.
3.3. Effect of melatonin and mifepristone on serum urea, uric acid, and potassium level
3.7. Effect of melatonin and mifepristone on oxidative stress parameters
A significant increase in serum urea level was observed in IRI group compared with control group. The treatment with melatonin resulted in a significant decrease in urea level compared with IRI group. The IRI þ melatonin þ mifepristone group demonstrated significant increase in serum urea level compared with melatonin low as well as high dose treatment. Similar pattern of results was observed with serum uric acid and serum potassium level (Table 1).
A significant increase in MPO activity was observed in IRI group compared with control group. The melatonin treatment significantly decreased the MPO activity compared with IRI group. The IRI þ melatonin þ mifepristone group demonstrated significant increase in MPO activity compared with IRI þ melatonin low as well as high dose group (Table 2). A significant increase in lipid peroxidation measured in terms of TBARS was observed in IRI group compared with control group. The melatonin treatment significantly decreased the TBARS level compared with IRI group. There was a significant rise in TBARS level in IRI þ melatonin þ mifepristone group compared with IRI þ melatonin high dose group (Table 2). Similar pattern of results was observed in SAG measured in terms of reduced NBT in the present study. A significant reduction in renal GSH level was observed in IRI group compared with control group. The melatonin treatment significantly increased the GSH level compared with IRI group. The IRI þ melatonin þ mifepristone group revealed significant reduction in GSH level compared with IRI þ melatonin low as well as high dose group. Similar pattern of results was observed in CAT activity between various groups used in the present study (Table 2).
3.4.
Effect of melatonin and mifepristone on FeNa
A significant increase in FeNa was observed in IRI group compared with control group. The treatment with melatonin resulted in a significant decrease in FeNa compared with IRI group. The IRI þ melatonin þ mifepristone group witnessed significant increase in FeNa compared with IRI þ melatonin high dose group (Table 1).
3.5. Effect of melatonin and mifepristone on LDH activity A significant increase in LDH activity was observed in IRI group compared with control group. The melatonin treatment significantly decreased the LDH activity, whereas LDH activity increased significantly in IRI þ melatonin þ mifepristone group compared with IRI þ melatonin low as well as high dose group (Table 1).
3.6. Effect of melatonin and mifepristone on microproteinuria A significant increase in level of urinary proteins was observed in IRI group compared with control group. The melatonin treatment significantly decreased level of urinary proteins. The IRI þ melatonin þ mifepristone group demonstrated significant increase in microproteinuria compared with IRI þ melatonin (4 and 10 mg/kg) group (Table 1).
3.8. Histopathologic evaluation of the effect of melatonin and mifepristone on renal tissues The hematoxylin and eosin staining revealed detachment of glomeruli from basement membrane, increased interstitial spaces, cell debris in tubules and neutrophil accumulation in renal sections of IRI group compared with control group. The treatment with melatonin resulted in reversal of IRIinduced changes in renal tissues. However, the treatment with mifepristone abolished melatonin-mediated renoprotective effect (Fig).
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520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584
Table 2 e Effect of melatonin and mifepristone on renal oxidative stress parameters. Groups Parameters MPO (U/g of tissue) TBARS (nM/mg of protein) SAG (pM/min/mg of tissue) GSH (mM/mg of protein) CAT (mM of H2O2 oxidized/min/mg of protein)
Control
Sham
IRI
IRI þ melatonin (4 mg/kg)
IRI þ melatonin (10 mg/kg)
IRI þ melatonin þ mifepristone
0.50 0.10 0.12 0.01
0.52 0.08 0.15 0.01
1.62 0.22a 0.46 0.02a
0.70 0.15b 0.26 0.03b
0.57 0.12b 0.25 0.01b
1.47 0.26a,c,d 0.39 0.04a,d
27.31 1.32
28.43 2.54
43.71 3.67a
30.05 2.14b
25.33 1.57b
3.49 0.28 0.39 0.02
3.14 0.42 0.37 0.03
1.81 0.16a 0.06 0.01a
2.91 0.32b 0.35 0.02b
3.11 0.41b 0.37 0.05b
40.27 2.33a,d 1.71 0.23a,c,d 0.11 0.02a,c,d
Values are expressed as mean standard error of the mean. a ¼ P < 0.05 versus control; b ¼ P < 0.05 versus IRI; c ¼ P < 0.05 versus IRI þ melatonin (4 mg/kg); d ¼ P < 0.05 versus IRI þ melatonin (10 mg/kg) group.
4.
Discussion
Various pharmacologic strategies used for management of AKI include use of loop diuretics that prevent reabsorption of water and reduces volume overload, dopamine that increases renal blood flow by splanchnic vasodilatation thereby increasing cardiac output and reperfusion pressure, fenoldopam that acts by increasing renal blood flow, natriuretic peptides to maintain pressure and homeostasis by modulating cardiac and renal function, atrial natriuretic peptide associated with an increase in glomerular filtration and Nacetylcysteine that is an antioxidant with vasodilating properties [27e31]. The IRI involves temporary cessation of blood flow to an organ followed by its resumption. The oxidative stress is caused by generation of reactive oxygen species (ROS) during ischemiaereperfusion [32,33]. It is an autocatalytic process that involves oxidative destruction of cellular membrane and accumulation of inflammatory mediators that lead to cell death [34]. The renal ischemiaereperfusion is the leading
cause of AKI and is associated with a high rate of mortality despite of the advancements in therapy. Various surgical procedures such as renal transplantation, partial nephrectomy, and aortic aneurysm involve transient discontinuation of renal blood flow. The resumption of the blood flow may lead to IRI thus introducing significant postoperative renal complications. The IRI-induced AKI is characterized by tubular damage and cell necrosis associated with ROS and accumulation of inflammatory cells [35,36]. Melatonin is a hormone secreted by pineal gland and is mainly responsible for controlling circadian rhythm. Apart from this, the role of melatonin has been explored in large number of pathophysiological processes such as regulation of immunomodulatory cytokines, inhibition of apoptosis, oncostatic action, management of cardiovascular complications including atherosclerosis, hypertension, angina pectoris, and neurologic disorders such as epilepsy and migraine [11]. Clinically, a decrease in melatonin rhythm with advancement of renal dysfunction has been observed in patients with chronic kidney disease [37]. The melatonin treatment demonstrated protection against microproteinuria in diabetic patients [38].
Fig e Effect of melatonin and mifepristone on renal tissues observed with hematoxylineeosin staining at 3400 magnification. ([A] control; [B] IRI; [C] IRI D melatonin [4 mg/kg]; [D] IRI D melatonin [10 mg/kg]; [E] IRI D melatonin D mifepristone). The arrows denote increased interstitial spaces, “*” denotes cell debris in tubules, “#” denotes detachment of glomeruli from basement membrane, whereas “**” denotes inflammatory cells. (Color version of figure is available online). 5.2.0 DTD YJSRE12693_proof 30 April 2014 8:36 pm ce
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Further preclinical and clinical studies are warranted to explore molecular mechanisms of melatonin-mediated renoprotection and its benefits in patients with renal dysfunction, respectively. The present study explored the involvement of progesterone receptors in melatonin-mediated protection against renal IRI. The melatonin is well documented to protect against IRI of various organs including heart [39], brain [40], and liver [41]. The correction of serum, urinary, and tissue oxidative stress parameters observed with melatonin treatment in the present study is in accordance with earlier studies [12,42]. The reduction in MPO activity along with reduction in neutrophil accumulation observed in hematoxylineeosin staining indicated anti-inflammatory property of melatonin. Being an amphiphilic molecule, it is able to penetrate and scavenge the cells [42]. The replenishment of reduced glutathione and increased CAT activity with melatonin treatment is important as depleted endogenous defense mechanisms are reported to produce more damage than increased levels of free radicals [20]. The melatonin (10 mg/kg) administered through various routes is reported to be protective against renal IRI in various studies [43e45]. No significant difference between two doses of melatonin (4 and 10 mg/kg) was observed in the present study that indicates that 4 mg/kg is effective dose and it could be used in future studies rather than using 10 mg/kg. The protective effect of melatonin was abolished with prior administration of progesterone receptor antagonist. There are various reports indicating cross talk between progesterone and melatonin [21,22]. The progesterone is a hormone required for maintenance of reproductive functions such as ovulation and implantation in females and androgen biosynthesis in males. Apart from its role in reproductive physiology, the neuroprotective role of progesterone against cerebral IRI is reported [46,47]. The progesterone treatment is noted to reduce the level of excitatory amino acids. Moreover, it increases the level of inhibitory neurotransmitters such as gamma amino butyric acid in brain [48]. It is also noted to upregulate the number of gamma amino butyric acid receptors [49]. The progesterone is well documented to reduce ROS and inflammatory cytokines in various pathologic conditions. The presence of progesterone receptors on kidney indicates their role in regulation of renal function [50]. The study from our laboratory indicated protective role of progesterone against renal IRI in dose-dependent manner [20]. The melatonin is reported to increase the progesterone levels [22] and also the expression of progesterone receptors [21,24]. The present study did not observe significant increase in serum progesterone level with melatonin treatment in rats. The single administration of melatonin given to rats in present study may be insufficient to produce a significant increase in progesterone level as observed with other agents such as ascorbic acid [20]. Moreover, most of the studies reporting increase in progesterone level have been conducted in cell cultures where cells are exposed to high concentrations of melatonin [51]. Third, the studies are carried out in granulosa or follicular cells that are specialized cells to produce progesterone compared with male rats used in the present study [23,51,52]. However, the treatment with mifepristone totally abolished melatonin-mediated antioxidant and protection against renal IRI that clearly
indicates involvement of progesterone receptors in melatonin-mediated renoprotective effect. Hence, we conclude that melatonin exhibits antioxidant effect that accounts for its renoprotective effect. Moreover, the progesterone receptors are essentially involved in mediating protective role of melatonin against ischemia reperfusion induced AKI in rats.
Acknowledgment Authors’ contributions: A.P.S. conceived, designed, and supervised the project. J.S. performed pharmacologic treatments. J.S., T.K., and R.B. did biochemical and histologic studies. J.S. and A.P.S. analyzed the data and wrote the article. All authors approved the article before submission to journal.
Disclosure The authors do not have any conflict of interest.
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