| Renal dopamine, as an endogenous natriuretic hormone, is responsible for60%sodium excretion during sodium loading. In the renal proximal tubule, dopamine inhibits the activity of sodium transporters such as Na+-K+-ATPase, via stimulation of DIR, thus leading to natriuresis and dieresis. Studies have shown that DIR agonist failed to promote sodium excretion in diabetic rats, owing to the impaired experession and fuction of DIR, thereby the activity of sodium transporters increased, thus leading to sodium retention. The exact mechanism of impaired DIR expression and function in diabetic rats is remained to be fully elucidated. It is reported that decreased DIR was in brains from STZ or alloxan-induced diabetic rats, and renal DIR function can be restored by reducing oxidative stress.Advanced glycation end products (AGEs) formed by glycosylation and oxidation, are a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with free amino groups of proteins. The AGE formation is a common process of both glycated and oxidative process. AGEs accumulate in the kidney and kidney is susceptible to oxidative damage. Oxidative stress-related diseases such as diabetes, hypertension or aging are manifested by increased AGEs level in vivo. However it is noted that these diseases are accompamied by a reduced sodium excretion and sodium retention, which implies a link between AGEs and DIR expression and function. There seems to be evidence linking reduced Dl receptor expression and function with abnormal AGEs levels in diabetic rats. However, it is not known whether AGEs can reduced the renal Dl receptor expression and function in diabetic rats.Objective:To investigate the effects of AGEs on the expression and function of renal DIR in STZ-induced diabetic rats, and elucidate the mechanism of renal DIR dysfunction. Methods:1. Establishment of diabetic rats model.SPF Male Sprague-Dawley rats were injected low dosage streptozotocin (STZ,35mg/kg) by intraperitoneal injection for3weeks, once a week. Control rats were injected citrate buffer alone.72h after the final injection, plasma glucose levels were measured by glucometer using a blood sample from tail-vein of rat. Rats with blood sugar level higher than300mg/dL were considered as diabetic rats. Then the rats were randomly assigned into four groups in which each group consisted of eight rats. Group I received vehicle distilled water stands for normal control. Group II received vehicle distilled water serves as diabetic control. Group III received apocynin (APO group) orally at a dose of200mg/kg/d. Group IV received l00mg/kg/d aminoguanidine (AG group) orally after chemical diabetes. The rats were killed after5weeks of administration. They were placed in individual metabolic cages before death and24-hour urine was collected. Biochemical parameters such as serum creatinine, urea nitrogen and urine protein were evaluated by an automatic biochemical analyzer. In the end, renal tissues was fixed in10%formaldehyde fixative, paraffin embedded, sectioned, and stained with Hematoxylin and Eosin (HE) according to standard method.2. The effect of AGEs on sodium retention in diabetic rats.As the description of method1, diabetic rats were randomly assigned into four groups in which each group consisted of7-6rats. Before the rats were executed, they were placed in individual metabolic cages for two days and24-hour urine was collected for two times. The urine was centrifuged and the supernatant was obtained for detection. After the collection of urine, sodium loading experiment was carried out. The rats were given a1%NaCl solution for3days, and other feeding conditions remained unchanged. Then they were placed in metabolic cages again and24-hour urine was collected and the volume of urine was recorded. The urine was centrifuged and the supernatant was for detection. After the rats were sacrificed, serum was collected for detection. The urine and serum sodium concentrations were detected by an automatic biochemical analyzer. 3. The mechanism of AGEs inhibiting renal DIR function in diabetic rats.As the description of method1, the diabetic rats were assigned to control, model, APO group and AG group. The formation of AGEs in serum and renal cortex was assessed by their characteristic fluorescence with excitation at370nm and emission at440nm, using a microplate fluorometer. Superoxide anion production and NADPH oxidase activity in serum and renal cortex were measured using the lucigenin-ECL method with a low concentration (10μmol/l) of lucigenin. Immunoblotting of the DIR was performed as described in the literature. And the renal proximal tubular was isolated for cAMP and Na+-K+-ATPase activity assay.Results:1. The diabetic rat model was successfully established.The model and treatment groups rats’symptoms are energy cachexia, polydipsia, polyuria, lowness reaction, slow action, hair stand and lackluster. Serume glucose was≥300mmol/dL and maintained to the end of experiment. There was a significant increase in urea nitrogen, creatinine clearance and24h urinary protein in model rats. Renal slices of model group rats can be seen a majority of glomerulus increasing, swelling, expanding of capillary lumen andred blood cell depositing in it. Glomerular basement membrane (GBM) incrassation can be observed. A few of the tubules atrophy or dilataltion and the tubular epithelia cells were swelling. AG and APO treatments lessened renal hypertrophy, decreased urinary protein, indicating AG and APO may improve renal function. Therefore, AGEs may promote renal injury in diabetic rats by inducing oxidative stress.2. The mechanism of AGEs on sodium retention in diabetic rats.(1) Model group had significantly higher urine volume (P<0.01) and significantly decreased urine sodium concentration (P<0.01) compared with control group at base level. After3days of sodium loading, model rats had significantly higher urine volume, urine sodium concentration, serum sodium concentration than the baseline, displaying polydipsia, polyuria and sodium retention.(2) Urine volume, urine sodium concentration, urine potassium concentration tended to be higher in AG and APO groups than model group at base level, but there was not significantly different (P>0.05). After sodium loading, urine volume and urine sodium concentration in AG and APO group rats were significantly higher than model group (P<0.05), while the serum sodium concentration is higher than that model group and less than control group(P<0.05and P<0.05, respectively), which indicating that AG or APO treatment can ameliorate sodium retention in diabetic rats.3. The mechanism of AGEs inhibiting renal DIR function in diabetic rats.(1) AGEs level, superoxide anion and NADPH oxidase activity in serum and renal cortex from model group were significantly elevated compared with control group, which means amplified glycated and oxidative process. The DIR expression in renal cortex, cAMP level in renal proximal tubule were decreased in model group, while the Na+-K+-ATPase activity was increased in model rats. The Dl-like receptor agonist fenoldopam failed to stimulate cAMP in renal proximal tubule from model rats, indicating the defective DIR function.(2) AGEs level, superoxide anion and NADPH oxidase activity in serum and renal cortex were elevated by AG and APO treatment. AG and APO supplementation increased DIR expression and the baseline cAMP level, while Na+-K+-ATPase activity in renal proximal tubular can be reduced by AG and APO supplementation. AG and APO treatments restored the DIR function by increasing fenoldopalm simulated cAMP level in renal proximal tubule. Thus, AG and APO supplementation reduced AGEs level, lowered oxidative stress to restore DIR expression and DIR dysfunction, as a result, Na+-K+-ATPase activity was reduced.Conclusions:1. AGEs and oxidative stress are the main causes of diabetic renal injury. AG and APO treatment can improve the diabetic rats’renal function.2. AGEs promoted sodium retention by increasing oxidative stress in diabetic rats. AG and APO treatment might improve sodium retention in diabetic rats.3. AGEs inhibit the expression and function of renal DIR in diabetic rats by inducing oxidative stress, thus leading to sodium retention. |
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