Accepted Manuscript Title: BAY 11-7082 ameliorates diabetic nephropathy by attenuating hyperglycemia-mediated oxidative stress and renal inflammation via NF-B Pathway Author: Sambasiva Rao Kolati Eshvendar Reddy Kasala Lakshmi Narendra Bodduluru Jalandhar Reddy Mareddy Shravan Kumar Uppalapu Ranadeep Gogoi Chandana C. Baruah Mangala Lahkar PII: DOI: Reference:
S1382-6689(15)00036-8 http://dx.doi.org/doi:10.1016/j.etap.2015.01.019 ENVTOX 2191
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
18-10-2014 22-1-2015 28-1-2015
Please cite this article as: Kolati, S.R., Kasala, E.R., Bodduluru, L.N., Mareddy, J.R., Uppalapu, S.K., Gogoi, R., Baruah, C.C., Lahkar, M.,BAY 11-7082 ameliorates diabetic nephropathy by attenuating hyperglycemia-mediated oxidative stress and renal inflammation via NF-rmkappaB Pathway, Environmental Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.etap.2015.01.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ip t
BAY 11-7082 ameliorates diabetic nephropathy by attenuating hyperglycemia-mediated oxidative stress and renal inflammation via NF-κB Pathway Sambasiva Rao Kolati1a, Eshvendar Reddy Kasala1a*, Lakshmi Narendra Bodduluru1, Jalandhar Reddy Mareddy1, Shravan Kumar Uppalapu1, Ranadeep Gogoi2, Chandana C
cr
Baruah3, Mangala Lahkar1, 4 1
Contributed equally
ed
a
M
an
us
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research- Guwahati, Bhangagarh-781032, Assam, India. 2 Department of Biotechnology, National Institute of Pharmaceutical Education and ResearchGuwahati, Bhangagarh-781032, Assam, India 3 Department of Pharmacology and Toxicology, College of veterinary sciences, Khanapara-781022, Assam, India. 4 Departmentof Pharmacology, Gauhati Medical College and Hospital, Bhangagarh-781032, Assam, India.
Running Title: BAY 11-7082 ameliorates diabetic nephropathy by NF-κB inhibition.
pt
Type of article: Original article
*
Ac ce
Word counts For abstract: 254 For the text: 3320 (Excluding tables and references)
Corresponding author: Eshvendar Reddy Kasala Research Scholar, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati Bhangagarh-781032, Assam, India E-mail: [email protected] Mobile number: +919700820750.
1|Page
Page 1 of 20
1. Introduction: Diabetic pandemic is a major cause of mortality and morbidity worldwide. It is projected to affect approximately 438 million individuals worldwide by 2030 [1]. The most common pathological
ip t
features of diabetic complications observing in about half of the diabetic population are diabetic nephropathy, neuropathy, retinopathy and cardio-myopathy. Among these, diabetic nephropathy (DN) is one of the most frequent micro-vascular complications of diabetes mellitus; it is the
cr
primary cause of end-stage renal disease and a major cause of mortality and morbidity in patients with kidney diseases worldwide. DN is characterized by a series of renal structure abnormalities
us
including basement membrane thickening, mesangial expansion, glomerulusclerosis and tubulointerstitial fibrosis [2].
an
Despite the benefits derived from the current therapeutics for diabetic nephropathy, namely strict control of glucose, blood pressure and blockade of the renin-angiotensin system, these
M
approaches still offer imperfect protection against renal progression [3]. This imperfection points to the need for newer therapeutic agents that have potential to affect primary mechanisms contributing to the pathogenesis of diabetic nephropathy [4].Growing evidence highlights the
ed
significance of inflammatory cascade in the initiation and progression of diabetic nephropathy [5]. Therefore investigations into anti-inflammatory strategies may offer new strategies for treating this
pt
deliberate condition. Numerous human and animal studies have shown that raised macrophage infiltration, activation of adhesion molecules and inflammatory cytokines in the diabetic kidney in
Ac ce
a manner similar to other immunologic renal diseases [6-8]. Nuclear factor- kappa B (NF-κB) is a ubiquitous and renowned transcription factor responsible for regulating the expressions of genes that are involved in inflammatory pathways such as proinflammatory cytokines, chemokines and adhesion molecules [9]. Since NF-κB plays a pivotal role in the inflammatory process, it has been an essential and attractive therapeutic target for the management of various inflammatory diseases including diabetic nephropathy. Increasing evidence demonstrates that NF-κB is activated and contributes to macrophage infiltration in experimental models of diabetic kidney disease [10-12]. Furthermore, recent studies suggest that high blood glucose, proteinuria, angiotensin II and mechanical stretching contribute to NF-κB activation [13-15].
2|Page
Page 2 of 20
BAY 11-7082 is [3-(4-Methylphenylsulfonyl)-2-propenenitrile] an IκB phosphorylation inhibitor. (Figure 1) BAY 11-7082 is a selective inhibitor of IKK-β and it also irreversibly inhibits IKK-α phosphorylation which results in down regulation of the NF-κB activation. Literature reports have shown its pharmacological activities that include anticancer, neuroprotective, anti-inflammatory
ip t
and anti-diabetic effects [15, 16]. Previously it was reported that BAY 11-7082 has ameliorated experimentally induced diabetic neuropathy by suppressing the NF-κB and NF-κB regulated
cr
oxidative stress and neuro-inflammation in Sprague Dawley (SD) rats [17].
In this study we investigated the effect of BAY 11-7082, a NF-κB inhibitor, on diabetic
us
nephropathy under the hypothesis that inhibition of the NF-κB pathway may improve renal function through the modulation of hyperglycemia induced oxidative–nitrosative stress and
an
inflammatory cascade in the kidneys of streptozotocin (STZ) induced diabetic rats.
M
2. Materials and methods: 2.1 Chemicals
ed
BAY 11-7082 was purchased from Santa Cruz Biotechnology (California, USA). Streptozotocin and rat ELISA kits for estimation of TNF-α, IL-1β and IL-6 in tissue homogenate were purchased from Sigma Aldrich (St. Louis, MO, USA). ELISA kit for estimation of NF-κBP65 level in the
pt
tissue homogenate was purchased from QAYEE-BIO (Shanghai, China). Blood urea nitrogen, creatinine and albumin strips were purchased from IDEXX laboratories (Westbrook, Maine, USA).
Ac ce
All other chemicals used in the study were of analytical grade. 2.2 Animals
Male SD rats of 6-8 weeks old, weighing 230-250g were procured from Central Animal House Facility of the institute (NIPER-Guwahati). The animals were acclimatized for one week before the study and fed with normal pellet diet (Amrut rat feed, Pranav Agro Industries Ltd., Maharashtra, India) and water ad libitum. They were housed in polypropylene cages and maintained under standard laboratory conditions (temperature 24±1°C, relative humidity 55±5%, and 12-hour light/dark cycle). All the investigational procedures were approved by the institutional animal ethics committee of Gauhati Medical College. (IAEC approval no: MC/32/2013/32) 3|Page
Page 3 of 20
2.3 Induction of diabetes and experimental design Diabetes was induced to the rats by single intraperitoneal injection of streptozotocin (50 mg/kg of body weight) dissolved in 0.1M cold citrate buffer of pH 4.4. Tail vein blood glucose levels were estimated using SD CHECK blood glucose monitoring system (SD biosensor Inc. Korea) after 48
ip t
hours of STZ injection. Animals with blood glucose level greater than 250 mg/dl were considered as diabetic and further used for the study. STZ causes pancreatic β cell apoptosis and develops an
cr
experimental model for the type 1 diabetes mellitus. The experimental study groups consists of non-diabetic animals used as normal control group and drug control group, STZ induced diabetic
us
animals (blood glucose more than 250mg/dl) used as diabetic control group, and two groups of diabetic animals were treated with BAY 11-7082 (1mg/kg & 3mg/kg). Normal control and drug control rats were administered with vehicle citrate buffer and highest dose of BAY11-7082
an
respectively. (Figure 2) Treatment with BAY 11-7082 was started 4 weeks after STZ injection and was continued for next 4 weeks i.e end of the study period. At the end of the study, animals were
M
fasted overnight, anaesthetized and sacrificed by cervical decapitation immediately after the withdrawal of blood.
ed
2.4 Preparation of kidney homogenate
Kidney tissues were collected from all the experimental groups of rats and rinsed in ice cold saline.
pt
The kidneys were homogenized in 0.1M Tris HCL buffer using Teflon homogenizer. After complete homogenization it was centrifuged at 12000g for 30 min at 40C, supernatant was
Ac ce
collected and stored at -800C until used for biochemical estimations. 2.5 Assessment of renal dysfunction At the end of the 8thweek, rats were kept individually in metabolic cages for 24 hours to collect urine for the measurement of urine output and renal function. Renal function was measured by determining plasma and urine creatinine, blood urea nitrogen (BUN) and serum albumin using semi-auto-analyzer (IDEXX vet test analyzer). Creatinine clearance (Ccr) was computed using the following formula: Creatinine clearance [Ccr] (ml/min)= [Urea creatinine/Serum creatinine]×urine volume (ml). Plasma glucose levels were measured at the end of the experiment to investigate the effect of BAY 11-7082 on glucose.
4|Page
Page 4 of 20
2.6 Assessment of oxidative stress and nitrosative stress The malondialdehyde content, a good measure of lipid peroxidation, was measured in the form of thiobarbituric acid-reactive substances by the method of Okhawa et al. (1979) [18]. Glutathione (GSH) was determined by the method of Ellman (1959) [19]. It is based on the development of a
ip t
yellow color upon addition of 5, 5'-dithiobis-(2-nitrobenzoic acid) to samples having sulfhydryl groups. Finally, calculated the nM amount of –SH content per milligram of tissue using reduced glutathione as a standard. Superoxide dismutase (SOD) was assayed by using the commercially
cr
available kit from Sigma Aldrich (St. Louis, MO, USA). The renal nitric oxide level was indirectly measured by determining the nitrite level using a colorimetric method based on the Griess reaction.
us
The detection of total nitrite is then determined as a colored azo dye product of the Griess Reaction. Protein content in all samples was estimated by the method of Lowry et al (1951) [20]
an
using bovine serum albumin as a standard. 2.7 Rat IL-1β, IL-6, and TNF-α ELISA
M
The levels of proinflammatory cytokines (TNF-α, IL-1β and IL-6) in kidney homogenate of control and experimental groups were estimated by specific ELISA kits according to the
ed
manufacturer's instructions (Sigma Aldrich (St. Louis, MO, USA)). The concentration of cytokines was determined spectrophotometrically at 450 nm. Standard plots were constructed and the concentrations for unknown samples were calculated from the standard plot. The analysis was
Ac ce
pt
done according to the manufacturer's instructions. 2.8 Quantification of NF-kappa B p65 subunit The nuclear levels of p65 may correlate positively with the activation of NF-κB pathway. The NFκB/p65 Active ELISA (QAYEE –BIO (Shanghai, China)) kit was used to measure NF-κB free p65 in the cytoplasmic fraction. The analysis was done according to the manufacturer's instructions. The results were expressed as ng/mg protein. 2.9 Histological analysis A part of kidney from all groups was fixed in 10% buffered neutral formalin for one week at room temperature. Then tissues were dehydrated in a graded series of alcohol, cleaned in xylene and then embedded in paraffin. Tissue embedded paraffin blocks were sectioned into 4 μm thickness 5|Page
Page 5 of 20
using a rotary microtome. Sections were stained with haematoxylin–eosin and changes in the stained sections were then examined under a light microscope by a pathologist without prior knowledge of the groups.
ip t
2.10 Statistical analysis All the values are expressed as mean ± SEM (n=6). Statistical analysis was performed in Graph Pad Prism 5 software (San Diego, CA, USA) using one way analysis of variance (ANOVA)
cr
followed by Tukey’s test as a post hoc analysis. A value of p
S1382-6689(15)00036-8 http://dx.doi.org/doi:10.1016/j.etap.2015.01.019 ENVTOX 2191
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
18-10-2014 22-1-2015 28-1-2015
Please cite this article as: Kolati, S.R., Kasala, E.R., Bodduluru, L.N., Mareddy, J.R., Uppalapu, S.K., Gogoi, R., Baruah, C.C., Lahkar, M.,BAY 11-7082 ameliorates diabetic nephropathy by attenuating hyperglycemia-mediated oxidative stress and renal inflammation via NF-rmkappaB Pathway, Environmental Toxicology and Pharmacology (2015), http://dx.doi.org/10.1016/j.etap.2015.01.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ip t
BAY 11-7082 ameliorates diabetic nephropathy by attenuating hyperglycemia-mediated oxidative stress and renal inflammation via NF-κB Pathway Sambasiva Rao Kolati1a, Eshvendar Reddy Kasala1a*, Lakshmi Narendra Bodduluru1, Jalandhar Reddy Mareddy1, Shravan Kumar Uppalapu1, Ranadeep Gogoi2, Chandana C
cr
Baruah3, Mangala Lahkar1, 4 1
Contributed equally
ed
a
M
an
us
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research- Guwahati, Bhangagarh-781032, Assam, India. 2 Department of Biotechnology, National Institute of Pharmaceutical Education and ResearchGuwahati, Bhangagarh-781032, Assam, India 3 Department of Pharmacology and Toxicology, College of veterinary sciences, Khanapara-781022, Assam, India. 4 Departmentof Pharmacology, Gauhati Medical College and Hospital, Bhangagarh-781032, Assam, India.
Running Title: BAY 11-7082 ameliorates diabetic nephropathy by NF-κB inhibition.
pt
Type of article: Original article
*
Ac ce
Word counts For abstract: 254 For the text: 3320 (Excluding tables and references)
Corresponding author: Eshvendar Reddy Kasala Research Scholar, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati Bhangagarh-781032, Assam, India E-mail: [email protected] Mobile number: +919700820750.
1|Page
Page 1 of 20
1. Introduction: Diabetic pandemic is a major cause of mortality and morbidity worldwide. It is projected to affect approximately 438 million individuals worldwide by 2030 [1]. The most common pathological
ip t
features of diabetic complications observing in about half of the diabetic population are diabetic nephropathy, neuropathy, retinopathy and cardio-myopathy. Among these, diabetic nephropathy (DN) is one of the most frequent micro-vascular complications of diabetes mellitus; it is the
cr
primary cause of end-stage renal disease and a major cause of mortality and morbidity in patients with kidney diseases worldwide. DN is characterized by a series of renal structure abnormalities
us
including basement membrane thickening, mesangial expansion, glomerulusclerosis and tubulointerstitial fibrosis [2].
an
Despite the benefits derived from the current therapeutics for diabetic nephropathy, namely strict control of glucose, blood pressure and blockade of the renin-angiotensin system, these
M
approaches still offer imperfect protection against renal progression [3]. This imperfection points to the need for newer therapeutic agents that have potential to affect primary mechanisms contributing to the pathogenesis of diabetic nephropathy [4].Growing evidence highlights the
ed
significance of inflammatory cascade in the initiation and progression of diabetic nephropathy [5]. Therefore investigations into anti-inflammatory strategies may offer new strategies for treating this
pt
deliberate condition. Numerous human and animal studies have shown that raised macrophage infiltration, activation of adhesion molecules and inflammatory cytokines in the diabetic kidney in
Ac ce
a manner similar to other immunologic renal diseases [6-8]. Nuclear factor- kappa B (NF-κB) is a ubiquitous and renowned transcription factor responsible for regulating the expressions of genes that are involved in inflammatory pathways such as proinflammatory cytokines, chemokines and adhesion molecules [9]. Since NF-κB plays a pivotal role in the inflammatory process, it has been an essential and attractive therapeutic target for the management of various inflammatory diseases including diabetic nephropathy. Increasing evidence demonstrates that NF-κB is activated and contributes to macrophage infiltration in experimental models of diabetic kidney disease [10-12]. Furthermore, recent studies suggest that high blood glucose, proteinuria, angiotensin II and mechanical stretching contribute to NF-κB activation [13-15].
2|Page
Page 2 of 20
BAY 11-7082 is [3-(4-Methylphenylsulfonyl)-2-propenenitrile] an IκB phosphorylation inhibitor. (Figure 1) BAY 11-7082 is a selective inhibitor of IKK-β and it also irreversibly inhibits IKK-α phosphorylation which results in down regulation of the NF-κB activation. Literature reports have shown its pharmacological activities that include anticancer, neuroprotective, anti-inflammatory
ip t
and anti-diabetic effects [15, 16]. Previously it was reported that BAY 11-7082 has ameliorated experimentally induced diabetic neuropathy by suppressing the NF-κB and NF-κB regulated
cr
oxidative stress and neuro-inflammation in Sprague Dawley (SD) rats [17].
In this study we investigated the effect of BAY 11-7082, a NF-κB inhibitor, on diabetic
us
nephropathy under the hypothesis that inhibition of the NF-κB pathway may improve renal function through the modulation of hyperglycemia induced oxidative–nitrosative stress and
an
inflammatory cascade in the kidneys of streptozotocin (STZ) induced diabetic rats.
M
2. Materials and methods: 2.1 Chemicals
ed
BAY 11-7082 was purchased from Santa Cruz Biotechnology (California, USA). Streptozotocin and rat ELISA kits for estimation of TNF-α, IL-1β and IL-6 in tissue homogenate were purchased from Sigma Aldrich (St. Louis, MO, USA). ELISA kit for estimation of NF-κBP65 level in the
pt
tissue homogenate was purchased from QAYEE-BIO (Shanghai, China). Blood urea nitrogen, creatinine and albumin strips were purchased from IDEXX laboratories (Westbrook, Maine, USA).
Ac ce
All other chemicals used in the study were of analytical grade. 2.2 Animals
Male SD rats of 6-8 weeks old, weighing 230-250g were procured from Central Animal House Facility of the institute (NIPER-Guwahati). The animals were acclimatized for one week before the study and fed with normal pellet diet (Amrut rat feed, Pranav Agro Industries Ltd., Maharashtra, India) and water ad libitum. They were housed in polypropylene cages and maintained under standard laboratory conditions (temperature 24±1°C, relative humidity 55±5%, and 12-hour light/dark cycle). All the investigational procedures were approved by the institutional animal ethics committee of Gauhati Medical College. (IAEC approval no: MC/32/2013/32) 3|Page
Page 3 of 20
2.3 Induction of diabetes and experimental design Diabetes was induced to the rats by single intraperitoneal injection of streptozotocin (50 mg/kg of body weight) dissolved in 0.1M cold citrate buffer of pH 4.4. Tail vein blood glucose levels were estimated using SD CHECK blood glucose monitoring system (SD biosensor Inc. Korea) after 48
ip t
hours of STZ injection. Animals with blood glucose level greater than 250 mg/dl were considered as diabetic and further used for the study. STZ causes pancreatic β cell apoptosis and develops an
cr
experimental model for the type 1 diabetes mellitus. The experimental study groups consists of non-diabetic animals used as normal control group and drug control group, STZ induced diabetic
us
animals (blood glucose more than 250mg/dl) used as diabetic control group, and two groups of diabetic animals were treated with BAY 11-7082 (1mg/kg & 3mg/kg). Normal control and drug control rats were administered with vehicle citrate buffer and highest dose of BAY11-7082
an
respectively. (Figure 2) Treatment with BAY 11-7082 was started 4 weeks after STZ injection and was continued for next 4 weeks i.e end of the study period. At the end of the study, animals were
M
fasted overnight, anaesthetized and sacrificed by cervical decapitation immediately after the withdrawal of blood.
ed
2.4 Preparation of kidney homogenate
Kidney tissues were collected from all the experimental groups of rats and rinsed in ice cold saline.
pt
The kidneys were homogenized in 0.1M Tris HCL buffer using Teflon homogenizer. After complete homogenization it was centrifuged at 12000g for 30 min at 40C, supernatant was
Ac ce
collected and stored at -800C until used for biochemical estimations. 2.5 Assessment of renal dysfunction At the end of the 8thweek, rats were kept individually in metabolic cages for 24 hours to collect urine for the measurement of urine output and renal function. Renal function was measured by determining plasma and urine creatinine, blood urea nitrogen (BUN) and serum albumin using semi-auto-analyzer (IDEXX vet test analyzer). Creatinine clearance (Ccr) was computed using the following formula: Creatinine clearance [Ccr] (ml/min)= [Urea creatinine/Serum creatinine]×urine volume (ml). Plasma glucose levels were measured at the end of the experiment to investigate the effect of BAY 11-7082 on glucose.
4|Page
Page 4 of 20
2.6 Assessment of oxidative stress and nitrosative stress The malondialdehyde content, a good measure of lipid peroxidation, was measured in the form of thiobarbituric acid-reactive substances by the method of Okhawa et al. (1979) [18]. Glutathione (GSH) was determined by the method of Ellman (1959) [19]. It is based on the development of a
ip t
yellow color upon addition of 5, 5'-dithiobis-(2-nitrobenzoic acid) to samples having sulfhydryl groups. Finally, calculated the nM amount of –SH content per milligram of tissue using reduced glutathione as a standard. Superoxide dismutase (SOD) was assayed by using the commercially
cr
available kit from Sigma Aldrich (St. Louis, MO, USA). The renal nitric oxide level was indirectly measured by determining the nitrite level using a colorimetric method based on the Griess reaction.
us
The detection of total nitrite is then determined as a colored azo dye product of the Griess Reaction. Protein content in all samples was estimated by the method of Lowry et al (1951) [20]
an
using bovine serum albumin as a standard. 2.7 Rat IL-1β, IL-6, and TNF-α ELISA
M
The levels of proinflammatory cytokines (TNF-α, IL-1β and IL-6) in kidney homogenate of control and experimental groups were estimated by specific ELISA kits according to the
ed
manufacturer's instructions (Sigma Aldrich (St. Louis, MO, USA)). The concentration of cytokines was determined spectrophotometrically at 450 nm. Standard plots were constructed and the concentrations for unknown samples were calculated from the standard plot. The analysis was
Ac ce
pt
done according to the manufacturer's instructions. 2.8 Quantification of NF-kappa B p65 subunit The nuclear levels of p65 may correlate positively with the activation of NF-κB pathway. The NFκB/p65 Active ELISA (QAYEE –BIO (Shanghai, China)) kit was used to measure NF-κB free p65 in the cytoplasmic fraction. The analysis was done according to the manufacturer's instructions. The results were expressed as ng/mg protein. 2.9 Histological analysis A part of kidney from all groups was fixed in 10% buffered neutral formalin for one week at room temperature. Then tissues were dehydrated in a graded series of alcohol, cleaned in xylene and then embedded in paraffin. Tissue embedded paraffin blocks were sectioned into 4 μm thickness 5|Page
Page 5 of 20
using a rotary microtome. Sections were stained with haematoxylin–eosin and changes in the stained sections were then examined under a light microscope by a pathologist without prior knowledge of the groups.
ip t
2.10 Statistical analysis All the values are expressed as mean ± SEM (n=6). Statistical analysis was performed in Graph Pad Prism 5 software (San Diego, CA, USA) using one way analysis of variance (ANOVA)
cr
followed by Tukey’s test as a post hoc analysis. A value of p
