ß-Endosulfan-mediated induction of pro-fibrotic markers in renal (HK-2) cells in vitro: A new insight in the pathogenesis of chronic kidney disease of unknown etiology.

Chronic kidney disease of unknown etiology (CKDu), manifested clinically as tubulo interstitial fibrosis, has emerged as the second major cause of chronic kidney disease (CKD) in the Indian subcontinent and various agrochemicals have been implicated in its occurance. Among the agrochemicals organochlorine pesticides particularly endosulfan is well known for its toxicity and recent residue analysis have shown its presence in the blood samples of general population. In this present study, we have investigated the consequences of endosulfan exposure at a concentration (0.01 µM) equivalent to their highest reported presence in human blood sample of some CKDu patients, to human renal proximal tubular epithelial (HK-2) cell line with regard to ROS generation and expression of profibrotic and epithelial to mesenchymal (EMT) markers in order to find out endosulfan's ability to induce profibrotic changes in renal cell. We demonstrated a significant increase in intracellular ROS generation and increased expression of TGF-ß1 when cells were incubated with ß-endosulfan (0.01 µM) indicating occurrence of oxidative stress and fibrotic process. Again, decreased expression of epithelial marker E-cadherin and increase in the expression of mesenchymal marker α-smooth muscle actin (α-SMA) suggest possible onset of EMT process. Pre-treatment with 5 mM concentration of anti-oxidant N-acetyl cysteine partially attenuated the above process. In conclusion, these findings suggest possible involvement of ß-endosulfan in the development of CKDu through oxidative stress and profibrotic signaling.

High levels of organochlorine pesticides in drinking water as a risk factor for type 2 diabetes: A study in north India.

Organochlorine pesticides (OCPs) are well known synthetic pesticides widely used in agricultural practices and public health program. Higher toxicity, slow degradation, and bioaccumulation are the significant challenges of OCPs. Due to its uses in agricultural and public health, contamination of drinking water and water table also increases day by day. Contaminated drinking water has become a significant issue and alarming signal for public health globally. The purpose of this study was to assess the recent trend of organochlorine pesticides (OCPs) level in drinking water and blood samples of the North Indian population and also to find out its association with glucose intolerance, lipid metabolism, and insulin resistance, which are known risk factors of type 2 diabetes mellitus (T2DM). A case-control study was conducted on 130 Non-Glucose intolerance (NGT), 130 pre-diabetes and 130 recently diagnosed T2DM subjects of the age group of 30-70 years. Patients consuming drinking water from the same source for at least ten years were included in this study for blood and water samples collection. Significantly higher levels of α-HCH, ß-HCH, γ-HCH, p,p'-DDE, and o,p'-DDT were found in groundwater samples. However, in tap water samples, the level of α-HCH was found to be slightly higher than the permissible limit of 0.001. Among all recruited subjects consuming contaminated groundwater, 42% had T2DM, 38% pre-diabetes, and the remaining 20% were found normal. We also observed that OCP contamination in groundwater is higher than tap and filter water. The levels of ß-HCH, p,p'-DDE, and o,p'-DDT were higher in the pre-diabetes and T2DM group than the NGT group. With an increase of OCPs level in groundwater, the blood OCPs level tends to increase T2DM risk. It depicts that the elevated OCPs level in consumed groundwater may contribute to increased risk for the development of T2DM after a certain period of exposure.

Lead-induced DNA damage and cell apoptosis in human renal proximal tubular epithelial cell: Attenuation via N-acetyl cysteine and tannic acid.

This study investigates the exposure of lead-induced reactive oxygen species (ROS) generation, DNA damage, and apoptosis and also evaluates the therapeutic intervention using antioxidants in human renal proximal tubular cells (HK-2 cells). Following treatment of HK-2 cells with an increasing concentration of lead nitrate (0-50 µM) for 24 h, the intracellular ROS level increased whereas the GSH level decreased significantly in a dose-dependent manner. Comet assay results revealed that lead nitrate showed the ability to increase the levels of DNA strand breaks in HK-2 cells. Lead exposure also induced apoptosis through caspase-3 activation at 30 µg/mL. Pretreatment with N-acetylcysteine (NAC) and tannic acid showed a significant ameliorating effect on lead-induced ROS, DNA damage, and apoptosis. In conclusion, lead induces ROS, which may exacerbate the DNA damage and apoptosis via caspase-3 activation. Additionally, supplementation of antioxidants such as NAC and tannic acid may be used as salvage therapy for lead-induced DNA damage and apoptosis in an exposed person.

Organochlorine pesticide level in patients with chronic kidney disease of unknown etiology and its association with renal function.

BACKGROUND: Involvement of agrochemicals have been suggested in the development of chronic kidney disease of unknown etiology (CKDu). The association between CKDu and blood level of organochlorine pesticides (OCPs) in CKDu patients has been examined in the present study. METHODS: All the recruited study subjects (n = 300) were divided in three groups, namely, healthy control (n = 100), patients with chronic kidney disease of unknown etiology (n = 100), and patients with chronic kidney disease of known etiology (CKDk) (n = 100). Blood OCP levels of all three study groups were analyzed by gas chromatography. RESULTS: Increased level of OCPs, namely α-HCH, aldrin, and ß-endosulfan, were observed in CKDu patients as compared to healthy control and CKD patients of known etiology. The levels of these pesticides significantly correlated negatively with the estimated glomerular filtration rate (eGFR) and positively with urinary albumin of CKD patients. Logistic regression analysis revealed association of γ-HCH, p, p'-DDE, and ß-endosulfan with CKDu on adjustment of age, sex, BMI, and total lipid content. CONCLUSIONS: Increased blood level of certain organochlorine pesticides is associated with the development of chronic kidney disease of unknown etiology.

Increased level of organochlorine pesticides in chronic kidney disease patients of unknown etiology: role of GSTM1/GSTT1 polymorphism.

Chronic kidney disease (CKD) of unknown etiology represents about 16% of CKD patients in Indian subcontinents and 10% worldwide. The aetiology of CKD of unknown etiology remains unclear though epidemiological studies indicate the involvement of the environmental toxins. Organochlorine pesticides (OCPs) have been detected in general population in India. It is possible that polymorphism of xenobiotic metabolizing enzymes (XMEs) may play an important role in this process. In this we intend to find out blood levels of OCPs in CKD patients of unknown etiology and to evaluate the consequence of glutathione S-transferase (GST) gene polymorphism on the same. We have assessed 270 CKD patients and 270 age-sex-matched healthy controls for this study. The blood OCP levels were analyzed by gas chromatograph. GSTM1, GSTT1 genotyping were carried out by multiplex PCR. Blood levels of HCH, endosulfan and total pesticides were significantly higher in CKD patients and negatively correlated with eGFR. The combined frequency of GSTM1(-)/GSTT1(-) genotype increased the risk of CKD by 1.8-fold as compared to healthy controls. To find out the dependence of blood OCPs level on genotype, we carried out logistic regression analysis and results revealed that GSTM1(-)/GSTT1(-) genotype associated significantly with a number of OCPs namely γ-HCH, p,p'-DDT and total pesticides. Polymorphism of XMEs not only increased accumulation of pesticides but also aggravates kidney dysfunction as evident from significant decrease in eGFR.

Association of CYP1A1 gene polymorphism with chronic kidney disease: a case control study.

CYP1A1 is an important xenobiotic metabolizing enzyme, present in liver and kidney. Expression of CYP1A1 enzyme increases manifold when kidney cells are exposed to nephrotoxins/chemicals leading to oxidative stress-induced cell damage. To study the association of CYP1A1 gene polymorphism in patients of chronic kidney disease with unknown etiology (CKDU), we recruited 334 CKDU patients and 334 age and sex matched healthy controls. CYP1A1*2A and *2C polymorphisms were studied by PCR-RFLP and allele specific-PCR respectively. Subjects carrying at least one mutant allele of CYP1A1*2A (TC, CC) and *2C (AG, GG) were shown to be associated with 1.4-2-fold increased risk of CKDU. Also, genotypic combinations of hetero-/homozygous mutants of CYP1A1*2A (TC, CC) with hetero-/homozygous mutant genotypes of CYP1A1*2C (AG, GG) i.e. TC/AG (p<0.01), TC/GG (p<0.05), CC/AG (p<0.05) and CC/GG (p<0.01) were associated with CKDU with an odd ratio ranging 1.8-3.3 times approximately. This study demonstrates association of CYP1A1 polymorphisms with CKDU.

A study on serum advanced glycation end products and its association with oxidative stress and paraoxonase activity in type 2 diabetic patients with vascular complications.

OBJECTIVES: Enhanced formation of advanced glycation end products (AGEs) formed secondary to hyperglycemic conditions has been linked to diabetes mellitus (DM) associated complications. We investigated the clinical relevance of estimating AGEs and their relationship with oxidative stress (OS) and paraoxonase (PON1) activity in type 2 DM (T2DM) in relation to development of vascular complications. DESIGN AND METHODS: Serum AGEs along with PON1 activity, protein carbonyl (PCO), advanced oxidation protein products (AOPP), lipid peroxidation (MDA), and total thiol (T-SH) were determined in 157 T2DM patients (DM without complications n=57, DM micro-vascular complications n=53, DM macro-vascular complications n=47) and 40 healthy controls. RESULTS: Serum AGE level increased significantly in various study groups in following manner: healthy control

Advanced glycation end products enhance reactive oxygen and nitrogen species generation in neutrophils in vitro.

Increased oxidative stress (OS) in diabetes mellitus is one of the major factors leading to diabetic pathology. However, the mediators and mechanism that provoke OS in diabetes is not fully understood, and it is possible that accumulation of advanced glycation end products (AGEs) formed secondary to hyperglycemic conditions may incite circulating polymorphonuclear neutrophils (PMN) to generate reactive oxygen species (ROS). In this report, we aim to investigate the effect of AGE on reactive oxygen and nitrogen species generation and subsequent OS in PMN. AGE-HSA exert dose- and time-dependent enhancement of ROS and reactive nitrogen intermediates (RNI) generation by PMN. Increased ROS and RNI generation were found to be mediated through the upregulation of NADPH oxidase and inducible nitric oxide synthase (iNOS), respectively, as evident from the fact that AGE-treated neutrophils failed to generate ROS and RNI in presence of diphenyleneiodonium, a flavoprotein inhibitor for both enzymes. Further increased generation of ROS and RNI ceased when the cells were incubated with anti-RAGE antibody suggesting the involvement of AGE-RAGE interaction. Also increased malondialdehyde (MDA) and protein carbonyl formation in AGE-exposed PMN suggest induction of OS by AGE. This study provides evidence that AGEs may play a key role in the induction of oxidative stress through the augmentation of PMN-mediated ROS and RNI generation and this may be in part responsible for development of AGE-induced diabetic pathology.