Background
Chronic hyperglycemic milieu of diabetes mellitus produces oxidative stress which is a key factor in the development of diabetic vascular complications such as diabetic nephropathy. Thus, in addition to the conventional treatments for intensive blood glucose control, it is essential to decrease oxidative stress to prevent the development of diabetic complications. NADPH oxidase has been identified as a major source of intrarenal
oxidative stress in the diabetic kidney. Thymol, a dietary monoterpene phenol found in Thymus species has displayed antioxidant, antiinflammatory, antiepileptic and neuroprotective activity.

Aim
The aim of the present study was to evaluate the effect of Thymol on serum advanced glycation end products (AGE), renal function biomarkers and renal oxidative stress in streptozotocin induced diabetic nephropathy in rats. In vitro NAPDH oxidase inhibitory activity of Thymol was evaluated in activated rat peritoneal macrophages to determine the possible mechanism of action of the antioxidant activity.

Method
Diabetes was induced in male wistar rats by single intraperitoneal injection of streptozotocin (45 mg/kg) dissolved in cold citrate buffer pH 4.5. Seven days after streptozotocin administration fasting blood glucose was estimated; rats with blood glucose levels greater than 13.88 mmol (>250 mg/dL) were termed diabetic and included in the study. Rats were randomly divided into six groups, namely: Group 1 (Normal
control group, n=6) received vehicle, Group 2 (Diabetic control, n=10) received vehicle, Group 3 (Standard control, n=10) received Metformin 500 mg/kg orally, Group 4 (Test group 1, n=10) received Thymol 1 mg/kg intraperitoneally, Group 5 (Test group 2, n=10)
received Thymol 5 mg/kg intraperitoneally, Group 6 (Test group 3, n=10) received Thymol 10 mg/kg intraperitoneally. Treatment was continued for period of four weeks. At the end of the study, rats were placed in metabolic cages for 24 hrs and urine was collected for
determination of urine protein and creatinine excretion. Blood was withdrawn from retroorbital plexus of 6 hours fasted rats; subsequently serum was separated and analysed for determination of serum albumin, serum creatinine and serum AGE. Thereafter, rats were perfused with normal saline and both kidneys were excised, blot dried and weighed. One half of each kidney was fixed in 10 % formalin solution for histopathological evaluation (Haematoxylin and Eosin staining, Periodic acid-Schiff base staining) and other half was homogenised for biochemical estimations. In vitro NADPH oxidase inhibitory activity was determined in resident macrophages obtained from peritoneal cavity
of rats injected with thioglycollate broth (3% w/v) 5 days prior to extraction of macrophages to induce an inflammatory response.Macrophages were incubated with rotenone (10 nM) to activate NADPH oxidase with or without different concentrations of Thymol (0.15 μg/ml-15 μg/ml) for 2 hours. Apocynin was used as standard control. The reduction of nitro blue tetrazolium to insoluble blue formazan was used to measure superoxide generation.

Results
Thymol dose dependently decreased kidney hypertrophy index (p<0.001) which was increased in the diabetic control group. Thymol at 5 mg/kg and 10 mg/kg significantly (p<0.001) increased serum albumin and decreased urine protein excretion, whereas, decreased serum creatinine and increased urine creatinine excretion. Serum AGE levels were significantly (p<0.01, 0.001) decreased by Thymol treatment at 5mg/kg and 10mg/kg. Thymol at 5mg/kg and 10mg/kg significantly (p<0.001) restored the antioxidant status in renal homogenate by increasing levels of reduced glutathione and antioxidant enzymes, namely superoxide dismutase, catalase and decreasing lipid peroxidation
and myeloperoxidase activity. Haematoxylin and Eosin staining demonstrated that diabetic control group exhibited severe morphological changes such as vacuolar degeneration, tubular collapse, hyalinisation of arterioles and scarring of tissues which was decreased in Thymol treated groups. The intensity of Periodic acid-Schiff base (PAS) staining of kidney was increased in diabetic control group whereas Thymol treatment significantly decreased the intensity of PAS staining. In vitro Thymol demonstrated marked NADPH oxidase inhibition of 72.5% at concentration of 1.5 μg/ml

Discussion
Thymol restored renal functions which were altered in the diabetic state. The above study demonstrates that Thymol exhibited protective effect on diabetic kidney by decreasing hyperglycemia induced oxidative stress. In vitro study indicated that Thymol possesses significant NAPDH oxidase inhibitory activity which was comparable with the standard inhibitor Apocynin. Thus the renoprotective effect of Thymol may be attributed to inhibition of renal NADPH oxidase. Further in vivo studies are warranted to elaborate the effect of Thymol on renal NAPDH oxidase.

Conflict of Interest
I have no potential conflict of interest to disclose

Thymol protects diabetic kidney by attenuating hyperglycemia induced oxidative stress