الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Proton pump inhibitors(PPIs) became one of the most widely prescribed classes of drugs in the last decade. Although this class of medication was initially considered safe, recent studies have shown an association between PPI use and long-term adverse effects. Among them, the possible association with kidney disease may be the most worrisome.
Diabetic nephropathy (DN) is the most common cause of end-stage kidney disease worldwide and is associated with increased morbidity and mortality in patients with both type1 and type2 diabetes. Recent evidence identified defective autophagy as a new culprit in the pathogenesis of DN. Autophagy is a highly conserved “self-eating” pathway by which cells degrade and recycle macromolecules and organelles and it serves as an essential mechanism to maintain homeostasis of glomeruli and tubules. Therefore, pharmacological modulation of autophagy may have an important impact on clinical outcome in diabetic renal disease. PPIs are among the drugs that are recently characterized as autophagy inhibitors. Accordingly, they may be hypothesized to adversly impact DN. Though renal side effects of PPIs have been documented in previous studies, their effect on DN are lacking.
In view of such consideration the present study was designed to evaluate the possible link between the use of the PPI: lansoprazole, renal autophagic changes and the progression of DN, in mice model of DN induced by streptozotocin (STZ) and high fat diet (HFD). The proposed effects of lansoprazole were analyzed in comparison to metformin and enalapril as an example of the standard therapy in DN.
The study was conducted on ninety male CD1 albino mice of body weight ranging from 20-25 g. The animals were randomly divided into 2 groups:
group Ι: Normal control group of 10 mice fed standard diet and received citrate buffer and gum acacia as vehicles for the same time and duration and by the same route as for diabetic mice.
group ΙΙ: Diabetic group: (80 mice) as type 2 DM was induced by feeding all animals with HFD for 2 weeks followed by i.p. injection of low dose STZ (55mg/kg) for five consecutive days. The occurrence of DM was confirmed by measuring blood glucose one week after final STZ injection. Only hyperglycemic animals with random blood glucose level above 280 mg/dl were further enrolled in the study and continued to be fed HFD till the end of the study. Four weeks after the last STZ injection, mice were randomly assigned into 6 groups to receive either drug treatment or vehicle for another 4 weeks as following: group ΙΙ-A, Diabetic control receiving daily oral administration of gum acacia.
group ΙΙ-B, Lansoprazole-treated diabetic mice receiving daily oral lansoprazole (15 mg/kg).
group ΙΙ-C, Metformin + Lansoprazole-treated diabetic mice receiving daily oral metformin (300 mg/kg) combined with daily oral lansoprazole (15 mg/kg).
group ΙΙ-D, Metformin-treated diabetic mice receiving daily oral metformin (300 mg/kg).
group ΙΙ-E, Enalapril + Metformin treated-diabetic mice receiving daily oral enalapril (0.5mg/kg), combined with daily oral Metformin (300 mg/kg).
group ΙΙ-F, Enalapril + Lansoprazole + Metformin-treated diabetic mice receiving daily oral enalapril (0.5mg/kg/day) combined with lansoprazole (15 mg/kg) and oral Metformin (300 mg/kg).
At different time points through-out the study, urine samples were taken to check albumin/creatinine ratio for confirmation of DN.
At the end of the 8th week, animals were put in metabolic cages for collection of 24 hours urine, for estimation of protein and creatinine in urine. Blood samples were collected after fasting for assessment of the following parameters: fasting blood glucose, glycosylated haemoglobin (HbA1c) level, serum creatinine, blood urea nitrogen (BUN), and creatinine clearance was calculated as an estimation of the glomerular filtration rate. Then one kidney was isolated, for assessment of renal tissue lipid peroxidation. The other kidney was fixed in paraffin for histopathological examination and for immunohistochemical detection of autophagy markers LC3 and P62.
The result of the present study showed that lansoprazole has a detrimental effect on DN, primarily presented as enhanced albuminuria, which is the most sensitive marker of podocytes injury. This was associated with enhanced oxidative stress reflected by high renal MDA. Also our findings revealed that administering lansoprazole alone to diabetic mice was associated with accumulation of both LC3II and P62 in both the renal and tubular cells, consistent with inhibition of autophagic flux, suggesting that inhibition of autophagy is one of the mechanisms of renal injurious effect of lansoprazole.
The metformin-treated groups showed better renal profile regarding the serum creatinine, BUN, albuminuria and glomerular filtration rate, together with decreased lipid peroxides. We demonstrated that metformin enhanced autophagic flux which, in addition to glycemic control, could possibly be one
of its renoprotective mechanisms. A significant deterioration of the renal functions and structure was observed in the lansoprazole-metformin treated group when compared to metformin alone. These changes were parallel to the detected changes in autophagic markers in terms of increased concentration of LC3-II positive cells in lansoprazole-metformin treated group versus metformin treatment alone. Likewise, the concentration of P62 positive cells was low with metformin treatment, and it was further increased when combined with lansoprazole. These findings indicate that lansoprazole partially opposes the observed reno-protective effects of metformin, most probably through autophagic blockade.
Another important finding is that the addition of enalapril to metformin did not prevent the deleterious effects of lansoprazole on the kidney. This is because the metformin+enalapril group showed the best renal profile including serum urea, creatinine, glomerular filtration rate and albuminuria comparative to all other groups. However, significant deterioration of all these renal markers was observed upon combination with lansoprazole.
Finally, regarding the metabolic parameters, our study proved that lansoprazole has no effect on glycemic control. The addition of lansoprazole to either metformin or metformin/enalapril was not only associated with worsened renal functions, but also with significantly higher levels of HBA1c, denoting less glycemic control on the long run.