Title: Role of imidazoline-1 receptor and inducible nitric oxide synthase (iNOS) in ethanol induced renal injury in normal and prediabetic rats. Introduction: Renal injury is a serious clinical problem that may be fatal if not properly treated. There are clear evidences that the occurrence of acute kidney injury is associated with early and late mortality, and also with high financial costs. One of the primary causes of renal injury is consumption of nephrotoxic agents as alcohol. Chronic alcoholism is a global health problem. A recent report by the world health organization found that roughly 3.3 million deaths or 5.9% of all global deaths are attributed to alcohol consumption. Chronic alcoholism impairs liver, kidney, pancreas and heart functions. The underlying mechanisms of alcohol induced nephrotoxicity include direct effects, like induction of renal oxidative stress, inflammatory and apoptotic pathways and indirect effects like hepatorenal syndrome and metabolic disorders. Some studies assumed that, all these effects contribute to the sudden loss of kidney functions precipitating acute kidney injury. However, more investigations and studies are needed to confirm these assumptions. Also, available treatments are not effective enough to completely manage the associated complications. Imidazoline-1 receptor (I1R) is a cell membrane receptor with a cytosolic subunit known as nischarin protein. Nischarin is responsible for the activation of I1R down-stream signals. Activation of I1R triggers the hydrolysis of phosphatidylcholine by activation of phospholipase A2 leading to synthesis of second messengers as arachidonic acid and subsequent generation of prostaglandins. Also, I1R mediates activation of insulin receptor substrate 4 enhancing the insulin action. Moreover, I1R mediates sympatho-inhibitory, cytoprotective and neuroprotective effects. Rilmenidine, a selective I1R agonist, reduces blood pressure by reacting with I1R in brain stem and kidney. Also, it improves glucose tolerance, insulin resistance and lipid profile in metabolic syndrome and type 2 diabetes. Inducible nitric oxide synthase (iNOS) is upregulated in alcohol induced kidney injury. It mediates production of nitric oxide that reacts with superoxides to form cytotoxic compounds like, peroxynitrites, also known as reactive nitrogen species (RNS). Aminoguanidine inhibits the formation of advanced glycation end products (AGEs) and reduces chronic diabetic complications through selective inhibition of iNOS. The current study focused on: • Investigating the deleterious effects of ethanol on kidney functions in normal and prediabetic rats. • Illustrating the therapeutic effects of selective and non-selective I1R agonists on ethanol induced renal injury in normal and prediabetic rats. • Illustrating the therapeutic effects of selective and non-selective iNOS inhibitors on ethanol induced kidney injury in normal and prediabetic rats. Experimental groups: Part I: Ethanol induced renal injury in normal rats: Eighty adult male albino rats weighting (180-200) gm were randomly divided into eight groups of 10 rats each as follow 1- Control (normal): rats received water. 2- Ethanol induced renal injury was performed by adding 20 ℅ (v/v) ethanol to daily drinking water for successive eight weeks. 3- Ethanol + Rilmenidine (I1R agonist) (3 mg/kg/day). 4- Ethanol + clonidine (adrenergic α2R/I1R agonist) (125 µg/kg/day). 5- Ethanol + efaroxan (I1R antagonist, 0.6 mg/kg/day) + clonidine. 6- Ethanol + phentolamine (α-blocker, 2 mg/kg/day) + clonidine. 7- Ethanol + aminoguanidine (iNOS inhibitor) (150 mg/kg/day) 8 - Ethanol + N(omega)-nitro-L-arginine methyl ester (L-NAME) (NOS inhibitor) (2mg/kg/day) * All drugs were dissolved in distilled water and administered orally in the last two weeks of ethanol administration period. Efaroxan and phentolamine were administered 30 minutes before clonidine administration. Part II: Ethanol induced renal injury in prediabetic rats: Sixty adult male albino rats weighting (100-120) gm were used and randomly divided into six groups of 10 rats each as follow: 1- Control (normal): rats received water. 2- Prediabetic ethanol fed rats: rats received fructose 12 weeks only, followed by ethanol for 8 weeks in the same concentrations mentioned previously. 3- Prediabetic ethanol fed rats +Rilmenidine (I1R agonist) (3 mg/kg/day). 4- Prediabetic ethanol fed rats + clonidine (adrenergic α2R/I1R agonist) (125 µg/kg/day). 5- Prediabetic ethanol fed rats + aminoguanidine (iNOS inhibitor) (150 mg/kg/day) 6 - Prediabetic ethanol fed rats + L-NAME (NOS inhibitor) (2mg/kg/day) * All drugs were dissolved in distilled water and administered orally in the last two weeks of ethanol administration period. At the end of treatments period, blood samples (followed by serum separation), urine samples and kidney tissue samples were collected for measurement of the following parameters: Blood samples Fasting blood glucose (FBG). Serum samples: Serum creatinine and urea. Serum insulin Serum advanced glycation end products (AGES) Serum (triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDLc), low density lipoprotein cholesterol (LDLc)) Urine samples: Urine albumin. Urine creatinine. Kidney samples: Kidneys were collected and rinsed thoroughly with saline, kidney tissues were used for biochemical and histopathological analysis. A small section of the kidney was immediately fixed in 10% neutral buffered formaldehyde for histopathological examination using (hematoxylin and eosin stain for cellular changes determination & Masson’s Trichrome stain for collagen deposition and fibrosis determination). The other parts of kidneys were collected, snap frozen in liquid nitrogen and stored at -80°C for further biochemical determination of the following parameters. 1) Renal oxidative stress parameters: superoxide dismutase (rSOD), malondialdehyde (rMDA), myeloperoxidase (rMPO) and total nitric oxide (rtNO). 2) Renal inflammatory and anti-inflammatory markers: tumor necrosis factor alpha (rTNF-α), interleukine-10 (rIL-10), Prostaglandin-E2 (rPGE2). 3) Renal Apoptotic marker (reactive Caspase-3) 4) Polymerase chain reaction (PCR) for determination of gene expression of nischarin subunit of I1R, phosphatidyl choline specific phospholipase-C (PC-PLC) and iNOS. The following results were obtained: Part I: 1) The effect of ethanol induced kidney injury in normal rats can be summarized as follow: Chronic ethanol ingestion significantly increased urinary albumin levels, FBG levels, serum levels of creatinine, urea, AGEs, TG, TC and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS and PC-PLC gene expression. Also, ethanol significantly increased renal fibrosis and cellular damage. On the other hand, ethanol significantly decreased urinary creatinine levels, serum insulin and HDLc levels and renal levels of SOD activity and IL-10. 2) Rilmenidine (3 mg/kg/day) effect on ethanol induced kidney injury in normal rats: Rilmenidine significantly decreased urinary albumin levels, serum levels of creatinine, urea, AGEs, TG, TC and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS gene expression. Also, rilmenidine treatment significantly decreased renal fibrosis and cellular damage. On the other hand, rilmenidine significantly increased urinary creatinine levels, serum HDLc and renal levels of PGE2, SOD activity, IL-10 and nischarin gene expression. 3) Clonidine (125μg/kg/ day) effect on ethanol induced kidney injury in normal rats: Clonidine significantly decreased urinary albumin levels, serum levels of creatinine, urea, AGEs, AST, ALT and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS gene expression. Also, clonidine treatment significantly decreased renal fibrosis and cellular damage. On the other hand, clonidine significantly increased urinary creatinine levels, serum insulin and HDLc and renal levels of PGE2, SOD activity, IL-10 and nischarin gene expression. On the other hand, blocking effect of clonidine using efaroxan significantly increased serum creatinine, urea and renal MDA level. 4) Aminoguanidine (150mg/kg/ day) effect on ethanol induced kidney injury in normal rats: Aminoguanidine significantly decreased urinary albumin levels, serum levels of creatinine, urea, AGEs and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS and PC-PLC gene expression. Also, aminoguanidine treatment significantly decreased renal fibrosis and cellular damage. On the other hand, aminoguanidine significantly increased urinary creatinine levels, serum HDLc and renal levels of PGE2, IL-10 and SOD activity. 5) L-NAME (2mg/kg/ day) effect on ethanol induced kidney injury in normal rats: L-NAME significantly decreased urinary albumin levels, serum levels of creatinine, urea, AGEs and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS and PC-PLC gene expression. Also, L-NAME treatment significantly decreased renal fibrosis and cellular damage. On the other hand, L-NAME significantly increased urinary creatinine levels, serum HDLc and renal levels of PGE2. Part II: 1) The effect of ethanol induced kidney injury in prediabetic rats can be summarized as follow: Chronic ethanol ingestion in prediabetic rats significantly increased urinary albumin levels, FBG levels, serum levels of creatinine, urea, AGEs, TG, TC and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS and PC-PLC gene expression. Also, ethanol significantly increased renal fibrosis and cellular damage. On the other hand, ethanol significantly decreased urinary creatinine levels, serum insulin and HDLc levels and renal levels of SOD activity and IL-10. 2) Rilmenidine (3 mg/kg/day) effect on ethanol induced kidney injury in prediabetic rats: Rilmenidine significantly decreased urinary albumin levels, FBG level, serum levels of creatinine, urea, insulin, AGEs, TG, TC and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS gene expression. Also, rilmenidine treatment significantly decreased renal fibrosis and cellular damage. On the other hand, rilmenidine significantly increased urinary creatinine levels, serum HDLc and renal levels of PGE2, SOD activity, IL-10 and nischarin gene expression. 3) Clonidine (125μg/kg/day) effect on ethanol induced kidney injury in prediabetic rats: Clonidine significantly decreased urinary albumin levels, FBG level, serum levels of creatinine, urea, insulin, AGEs, TG, TC and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS gene expression. Also, clonidine treatment significantly decreased renal fibrosis and cellular damage. On the other hand, clonidine significantly increased urinary creatinine levels, serum HDLc and renal levels of PGE2, SOD activity, IL-10 and nischarin gene expression. 4) Aminoguanidine (150mg/kg/ day) effect on ethanol induced kidney injury in prediabetic rats: Aminoguanidine significantly decreased urinary albumin levels, FBG level, serum levels of creatinine, urea, insulin, AGEs, TG, TC, and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS and PC-PLC gene expression. Also, aminoguanidine treatment significantly decreased renal fibrosis and cellular damage. On the other hand, aminoguanidine significantly increased urinary creatinine levels, serum HDLc and renal levels of PGE2, IL-10 and SOD activity. 5) L-NAME (2mg/kg/ day) effect on ethanol induced kidney injury in prediabetic rats: L-NAME significantly decreased urinary albumin levels, FBG level, serum levels of creatinine, urea, insulin, AGEs, TC and LDLc, and renal levels of MDA, MPO activity, tNO, TNF-a, caspase-3, iNOS and PC-PLC gene expression. Also, L-NAME treatment significantly decreased renal fibrosis and cellular damage. On the other hand, L-NAME significantly increased urinary creatinine levels and serum HDLc level. Based on the previous results, the following can be concluded:  The underlying mechanisms of ethanol induced kidney injury include direct activation of renal oxidative stress, inflammation and apoptosis in addition to metabolic impairments.  Ethanol induced kidney injury was more sever in prediabetic rats than normal rats.  Agonists of I1R (Rilmenidine & Clonidine) are promising new treatments that can ameliorate all pathological aspects of ethanol induced kidney injury.  iNOS inhibitors (aminoguanidine and L-NAME) can protect against ethanol induced kidney injury in normal and prediabetic rats, due to their antioxidant, anti-inflammatory, anti-apoptotic, and cytoprotective effects. Recommendations: This study recommends using I1R agonists and iNOS inhibitors, after fulfilling clinical requirements, for treatment of acute kidney injury cases with or without insulin resistance.