الفهرس 
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Abstract
This thesis is devoted to evaluate the potential role of gasotransmitters in the protective effect of the DPP-4 inhibitor; sitagliptin in DN. In addition. an attempt was undertaken to clarify the role of oxidative stress , nitrosative stress, inflammation and apoptosis in mediating the effects of sitagliptin and gasotransmitters. Type 2 diabetes was induced in overnight fasted rats by i.p. injection of 110 mg/kg nicotinamide (NA, 4% solution in saline) followed after 15 min by a single i.p. injection of 60 mg/kg streptozotocin (STZ) freshly prepared in 0.1 mol cold citrate buffer (pH 4.5). The STZ+NA treated animals were allowed to drink 5% glucose solution instead of drinking water for 24 h after STZ+NA challenge to overcome initial STZ-induced hypoglycemic mortality. Blood samples were collected from the tail vein of rats before and after 3 and 7 days of STZ+NA injection. Blood glucose levels were measured using a hand-held glucometer. Rats with random blood glucose levels more than 250 mg/dl on both days were considered as diabetic and used for diabetic nephropathy studies. Control animals were treated likewise with the citrate buffer. In preliminary experiments, two groups of diabetic animals, 8 rats each were used. Blood and urine samples were collected from the first group after 4 weeks of STZ+NA injection and from the second group after 8 weeks of STZ+NA injection. Blood and urine samples were also collected from two groups of control animals after 4 and 8 weeks of citrate buffer injection. In another set of experiments, the diabetic animals were divided after the 7th day of STZ+NA administration, into 8 groups, 8 rats each. Rats of group-I were treated with 10 mg/kg/day sitagliptin (0.5% solution in saline) orally for seven weeks. Group-II and III rats were treated for seven weeks with 10 mg/kg/day sitagliptin orally in combination with100 mg/kg/day L-arginine (L-A, 4% solution in saline) i.p. and 10 mg/kg/day L-N(G)-nitro arginine methyl ester (L-NAME, 1% solution in saline) i.p., respectively. Animals of group-IV and V were treated for seven weeks with 10 mg/kg/day sitagliptin orally in combination with 0.1 mg/kg/day carbon monoxide- releasing molecule-A1 (CORM-A1, 0.01% solution in saline) and 0.25 mg/kg/day zinc protoporphyrin (Znpp, 0.05% solution in saline) i.p., respectively. Rats of group-VI and VII were treated for seven weeks with 10 mg/kg/day sitagliptin orally in combination with 3 mg/kg/day sodium hydrosulfide (NaHS, 0.3% solution in saline) and 5 mg/kg/day DL-propargylglycine (DL-PAG, 0.5% solution in saline) i.p., respectively. Control animals were treated likewise with the normal saline. To collect urine samples from diabetic animals, each rat was housed individually in a metabolic cage for 24 h, one day before the end of each experimental duration. At the end of each experimental duration, the overnight fasted rats were sacrificed by decapitation. Blood and kidney tissues were obtained from each animal for biochemical measurements. Blood and urine samples were centrifuged for 10 min using Eppendorff centrifuge. After centrifugation, serum and urine samples were collected for estimation of urea, blood urea nitrogen , creatinine, creatinine clearance and albumin. The serum and urine samples could be used directly or stored at -80oC until assay. The isolated kidneys from each animal were rinsed in ice-cold saline. One kidney was kept in 10% formalin for histopathological and immunohistochemical studies and the other kidney was rinsed in ice-cold saline, dissected, cleaned from the fat and other tissues and blotted carefully. The kidney tissue was cut into small pieces and a suitable weight from them was homogenized in 10% w/v phosphate buffer (pH 7.4) or saline by using a motor-driven Teflon pestle.The homogenate was divided into 2 portions. The first portion was centrifuged for 10 min at 10,000 rpm and the supernatant was used for estimation of malondialdehyde (MDA), nitrite, tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), caspase-3, hydrogen sulfide (H2S) and heme oxygenase-1 (HO-1) levels directly or stored at -80oC until assay. To the second part of homogenate an equal volume of perchloric acid (1 mol/l) was added and mixed by vortexing. The mixture was allowed to stand for 5 min at room temperature. After centrifugation for 10 min, the supernatant was collected carefully and used for estimation of intracellular reduced glutathione (GSH) level directly or stored at -80oC until assay. Treatment of rats with 60 mg/kg STZ i.p. after 15 min of administration of 110 mg/kg NA i.p. produced a significant increase in the blood glucose levels after 3 days and one week as compared to control rats. Administration of STZ+NA to rats also produced a significant increase in the blood glucose levels after 4 and 8 weeks as compared to control rats. Treatment of animals with 110 mg/kg NA i.p. followed after 15 min by i.p injection of 60 mg/kg STZ produced after 4 weeks non-significant changes in the serum urea, blood urea nitrogen, urine urea, serum creatinine, urinary cratinine , creatinine clearance, serum albumin and urine albumin levels levels in comparison to control animals. The same treatment for the same period produced no changes in the kidney MDA, GSH, TNF-α , IL-1ß and caspase-3 levels as compared to control animals. Also, the same treatment produced after 4 weeks produced no changes in the renal nitrite, HO-1 and H2S levels. After 4weeks of treatment of animals with 110 mg/kg NA and 60 mg/kg STZ i.p. produced no histopathological changes in the kidney tissue. Similar treatment of animals with 110 mg/kg/day NA and 60 mg/kg/day STZ produced after 8 weeks a significant increase in the serum urea, blood urea nitrogen, serum creatinine and urine albumin (microalbuminuria) as well as a significant decrease in the urine urea, urine creatinine , creatinine clearance and serum albumin levels as compared to control animals. Similar treatment of rats produced after 8 weeks a significant increase in the kidney MDA , TNF-α, IL-1ß and caspase-3 levels but a significant decrease in the kidney intracellular GSH,. Also, similar treatment for the same duration produced a significant increase in the renal nitrite and a significant decrease in the renal HO-1 and H2S levels in comparison to control animals. After 8 weeks of treatment of animals with 110 mg/kg NA and 60 mg/kg STZ i.p., marked histopathological changes in the kidney tissue were observed. Daily administration of 10 mg/kg sitagliptin orally to diabetic rats for seven weeks produced a significant decrease in blood glucose levels after 4 and 8 weeks of induction of diabetes and significantly decreased the serum urea, blood urea nitrogen and increased urine urea levels as compared to STZ+NA treated animals. Also, administration of 10 mg/kg/day sitagliptin orally for 7 weeks to diabetic rats significantly decreased the serum creatinine and urine albumin (Microalbuminurea) meanwhile increased the urine creatinine, creatinine clearance and serum albumin levels. This treatment significantly produced a decrease in the renal MDA, TNF-α, IL-1ß and caspase-3 levels and an increase in the renal intracellular GSH levels. Administration of 10 mg/kg/day sitagliptin orally to diabetic rats for 7 weeks significantly decreased the renal nitrite and increased the renal HO-1and H2S levels in comparison to diabetic animals. Administration of 10 mg/kg/day sitagliptin orally for 7 weeks to diabetic rats ameliorated DN-induced histopathological changes of the kidney tissue. Co-administration of 10 mg/kg/day sitagliptin orally with100 mg/kg/day L-A i.p. to diabetic rats significantly increased the blood glucose levels after 4 and 8 weeks of induction of diabetes as compared to diabetic rats treated with sitagliptin only. Daily administration of 10 mg/kg sitagliptin orally concurrently with 100 mg/kg L-A i.p. for 7 weeks to diabetic rats significantly increased the serum urea, blood urea nitrogen, serum creatinine and urine albumin (microalbuminuria) levels but significantly decreased the urine urea, urine creatinine , creatinine clearance and serum albumin levels as compared to diabetic rats treated with sitagliptin only. The same treatment significantly increased the renal MDA, TNF-α, IL-1ß ,caspase-3 and nitrite and decreased the intracellular GSH levels and decreased the renal HO-1 and H2S levels in comparison to diabetic animals treated with sitagliptin for the same period. Co-administration of 10 mg/kg/day sitagliptin orally with 10 mg/kg/day L-NAME i.p. to diabetic rats for 7 weeks decreased the blood glucose levels after 4 and 8 weeks of induction of diabetes as compared to diabetic rats treated with sitagliptin only. Concurrent administration of 10 mg/kg/day sitagliptin orally with 10 mg/kg/day L-NAME i.p. for 7 weeks to diabetic rats significantly decreased the serum urea, blood urea nitrogen, serum creatinine and urine albumin (microalbuminuria) levels as well as increased the urine urea, urine creatinine , creatinine clearance and serum albumin levels in comparison to diabetic animals treated with sitagliptin for the same period.This treatment with sitagliptin and L-NAME also produced a significant decrease in the renal MDA, TNF-α, IL-1ß and caspase-3 levels and increase in the renal intracellular GSH levels Also, this treatment with sitagliptin and L-NAME produced a significant decrease in the renal nitrite and a significant increase in the renal HO-1 and H2S levels as compared to diabetic animals treated with sitagliptin for the same period. Daily administration of 10 mg/kg sitagliptin orally concurrently with 100 mg/kg L-A i.p. for 7 weeks to diabetic rats reduced the beneficial effects of sitagliptin on the DN-induced histopathological changes in the kidney tissue while daily administration of 10 mg/kg sitagliptin orally concurrently with 10 mg/kg L-NAME i.p. for the same period enhanced the beneficial effects of sitagliptin on the DN-induced histopathological changes in the kidney tissue. Concomitant administration of 10 mg/kg/day sitagliptin orally with 0.1 mg/kg/day CORM-A1 i.p. to diabetic rats for 7 weeks with significantly decreased the blood glucose levels after 4 and 8 weeks of induction of diabetes as compared to rats treated with sitagliptin only. Treatment of diabetic rats with sitagliptin and CORM-A1 for 7 weeks also produced a significant decrease in the serum urea, blood urea nitrogen, serum creatinine and urine albumin (microalbuminuria) as well as significant increase in urine urea, urine creatinine, creatinine clearance and serum albumin levels. Also, Administration of 10 mg/kg/day sitagliptin orally concomitantly with 0.1 mg/kg/day CORM- A1 i.p. to diabetic rats for 7 weeks also produced a significant decrease in the renal MDA, TNF-α, IL-1ß and caspase-3 levels as well as a significant increase in the intracellular GSH levels in comparison to diabetic animals treated with sitagliptin for the same period. Similar treatment of diabetic with sitagliptin and CORM-A1 significantly decreased the renal nitrite and increased the renal HO-1 and H2S levels in comparison to diabetic animals treated with sitagliptin for the same period. Administration of 10 mg/kg/day sitagliptin orally concurrently with 0.25 mg/kg/day Znpp i.p. to diabetic rats for 7 weeks significantly increased the blood glucose levels after 4 and 8 weeks of induction of diabetes in comparison to sitagliptin-treated diabetic animals for the same duration. Administration of 10 mg/kg/day sitagliptin orally concurrently with 0.25 mg/kg/day Znpp i.p. to diabetic rats for 7 weeks significantly increased the serum urea, blood urea nitrogen, serum creatinine and urine albumin (microalbuminuria) levels. However, this treatment with sitagliptin and Znpp decreased the urine urea, urine creatinine, creatinine clearance and serum albumin levels. Administration of 10 mg/kg/day sitagliptin orally concurrently with 0.25 mg/kg/day Znpp i.p. to diabetic rats for 7 weeks also produced a significant increase in the renal MDA, TNF-α, IL-1ß and caspase-3 levels and a significant decrease in the intracellular GSH levels in comparison to diabetic animals treated with sitagliptin only for the same period. The same co-treatment increased the renal nitrite and decreased the renal HO-1 and H2S levels in comparison to diabetic animals treated with sitagliptin only for the same period.
Daily administration of 10 mg/kg sitagliptin orally concurrently with 0.1 mg/kg CORM-A1 i.p. for 7 weeks to diabetic rats enhanced the beneficial effects of sitagliptin on DN-induced histopathological changes in the kidney tissue. Administration of 10 mg/kg/day sitagliptin orally concurrently with 0.25 mg/kg/day Znpp i.p. for the same period markedly antagonized the beneficial effects of sitagliptin on DN-induced histopathological changes in the kidney tissue. Co-administration of 10 mg/kg/day sitagliptin orally and 3 mg/kg NaHS i.p. to diabetic rats for 7 weeks significantly decreased the blood glucose levels after 4 and 8 weeks of induction of diabetes as compared to diabetic animals treated with sitagliptin only. Administration of 10 mg/kg/day sitagliptin orally concurrently with 3 mg/kg/day NaHS i.p. to diabetic rats for 7 weeks produced a significant decrease in the serum urea, blood urea nitrogen, serum creatinine and urine albumin (microalbuminuria) levels as well as a significant increase in the urine urea, urine creatinine, creatinine clearance and serum albumin levels. The same treatment of diabetic rats with sitagliptin and NaHS significantly decreased the renal MDA, TNF-α, IL-1ß and caspase-3 meanwhile, increased the intracellular GSH levels. This co-treatment produced a significant decrease in the renal nitrite and significant increase in the renal HO-1 and H2S levels in comparison to diabetic animals treated with sitagliptin for the same period. Concomitant administration of 10 mg/kg/day sitagliptin orally with 5 mg/kg DL-PAG i.p. to diabetic rats for 7 weeks significantly increased the blood glucose levels after 4 and 8 weeks of induction of diabetes in comparison to sitagliptin-treated diabetic animals for the same duration. Daily administration of 10 mg/kg/day sitagliptin orally concurrently with 5 mg/kg/day DL-PAG i.p. to diabetic rats for 7 weeks significantly increased the serum urea, blood urea nitrogen, serum creatinine and urine albumin (microalbuminuria) levels but decreased the urine urea, urine creatinine , creatinine clearance and serum albumin levels. The same treatment significantly increased the renal MDA, TNF-α, IL-1ß and caspase-3 and decreased the intracellular GSH levels. This co-treatment with sitagliptin and DL-PAG produced a significant increase in the renal nitrite and decreased the renal HO-1 and H2S levels in comparison to diabetic animals treated with sitagliptin for the same period. Daily administration of 10 mg/kg sitagliptin orally concurrently with 3 mg/kg NaHS i.p. for 7 weeks to diabetic rats potentiated the beneficial effect of sitagliptin on DN-induced histopathological changes in the kidney tissue. Co-administration of 10 mg/kg/day sitagliptin orally with 5 mg/kg/day DL-PAG i.p. for the same period antagonized the ameliorative effect of sitagliptin on the histopathological changes in the kidney tissue. In this study, the immunohistochemical analysis of the kidney tissue obtained from the rats with D.N showed a decrease in eNOS, HO-1 and CSE protein expressions but an increase in the protein expression of iNOS. Daily administration of 10 mg/kg sitagliptin orally concurrently with 10 mg/kg L-NAME, 0.1 mg/kg CORM-A1 or 3 mg/kg NaHS i.p. for 7 weeks to diabetic rats significantly increased the protein expressions of HO-1 and CSE in the kidney tissue. The protein expressions of iNOS was significantly reduced by administration of sitagliptin and sitagliptin with L-NAME, CORM-A1 or NaHS. The protein expressions of eNOS was significantly increased by administration of 10 mg/kg sitagliptin and sitagliptin with 0.1 mg/kg/day CORM-A1 or 3 mg/kg/day NaHS i.p. to diabetic rats for 7 weeks but significantly decreased by administration of 10 mg/kg sitagliptin and sitagliptin concurrently with 10 mg/kg/day L-NAME i.p. Co-administration of 10 mg/kg/day sitagliptin orally and 100 mg/kg/day L-A, 0.25 mg/kg/day ZnpPP or 5mg/kg/day DL-PAG i.p. for 7 weeks to diabetic rats significantly decreased the protein expressions of HO-1 and CSE in the kidney tissue. The protein expressions of iNOS was significantly increased by similar treatments for the same duration. Treatment of diabetic rats with 10 mg/kg/day sitagliptin orally in combination with100 mg/kg/day L-A increased protein expression of eNOS but co-administration of 10 mg/kg/day sitagliptin orally with 0.25 mg/kg/day Znpp or 5mg/kg/day DL-PAG i.p. decreased the protein expression of eNOS in the kidney tissue. Results of this work lead to the following conclusions: 1- Sitagliptin inhibited DN-induced impairment of renal function and structure. 2- Sitagliptin produced this protective effect due to, in addition to its glucose lowering effect, inhibition of DN-induced oxidative stress, nitrosative stress, inflammation, apoptosis, NO overproduction and decreased CO and H2S levels in the kidney tissue. 3- The inhibition of NO production and increasing the CO and H2S levels by L-NAME, CORM-A1 and NaHS potentiated the renoprotective effect of sitagliptin against DN. NO donor and CO and H2S biosynthesis inhibitors antagonized the beneficial effect of sitagliptin in DN. 4-Thus, there is an interrelationship between DN, sitagliptin and gasotransmitters. Also, gasotransmitters appear to play a pivotal role in the development of DN and in mediating the renoprotective effect of sitagliptin in DN.