| OBJECTIVE Diabetic nephropathy (DN) is one of the most serious microvascularcomplications of diabetes. Much work has been done, but the molecular mechanismsunderlying the pathogenesis of DN have not been fully elucidated. Berberine (BBR), aneffective compound of Chinese traditional herbal medicine, has preventive effects ondiabetes and its complications. Our previous study showed that BBR has a certain effecton G protein-adenylate cyclase (AC)-cAMP signaling pathway. Nevertheless, it remainsunclear whether BBR could modulate the expression of GRKs, a modulator of Gprotein-AC-cAMP signaling pathway, to prevent the progression of DN in rats or not?In our study, we attempted to investigate the renoprotective effects and its relativemechanisms of BBR in rats with high-fat diet and streptozotocin (STZ)-induced DNmodel.METHODS DN model in healthy male SD rats was induced with STZ onceintraperitoneal injection in a dose of35mg/kg after having high-fat diet for six weeks. After injected, all animals continued on the high-fat diet for72hours. The animals withfasting blood glucose(FBG) level of greater than or equal to11.1mmol/L and onlyuniformly diabetic rats were used in the study. Diabetic rats were randomly divided into7groups: diabetic rats without any drug treatment(HFDC); diabetic rats treated withBBR at a dose of50,100or200mg/kg every day; diabetic rats treated with Enalapril ata dose of1mg/kg; Xiaoke Wan(XKW) at a dose of800mg/kg and Simvastatin at a doseof2mg/kg every day served as positive control. Additionally, there were non-diabeticcontrol (NC) rats that neither received STZ nor the high-fat diet and high-fat dietcontrol (HFC) rats. Except NC, HFC and HFDC groups without any drug treatmentwere given an equal volume of vehicle (CMC-Na), while the other groups were givenintragastrically drugs for8weeks. The drug dose was adjusted according to the changeof animal weight. All the rats were allowed to continue to feed on their respective dietsuntil the end of the study. During the experiment, the rats’ diet, drinking and urinationsituation were observed and body weight was measured every week. FBG wasmeasured every2weeks on lateral tail vein blood samples. Eight weeks after theinduction of diabetes, all rats were housed individually within metabolic cages and urinewas collected from the rats housed in metabolic cages for24h. The overnight fastedrats were sacrificed. Blood samples were obtained from femoral artery, centrifuged,and the supernatant was used for measurement of glucose, lipid metabolic parametersand so on. Kidney weight to body weight (KW/BW) ratio, total urine protein for24hours to urine creatinine(UP/C)ratio, urine microalbumin for24hours to urinecreatinine(U.A/C), blood urea nitrogen (BUN), serum creatinine (SCr), high densitylipoprotein-cholesterol (HDL-c) and low density lipoprotein-cholesterol (LDL-c) weremeasured respectively. Separate kidney from the rats, HE staining and masson’strichrome staining were used to observe the pathological changes of kidney, andtransmission electron microscopy was used to observe glomerular ultrastructure. Fibronectin (FN), type IVcollagen (collagen IV), transforming growth factor-β1(TGF-β1)and connective tissue growth factor (CTGF)were detected by immunohistochemistry.The expression of GRKs in renal cortex fromDN rats were measured by westernblotting. The expression change of cAMP was investigated by radioimmunoassay. Thedifferences between these groups were compared.RESULTS1Effects of BBR treatment on FBG and BW of DN ratsFBG was significantly higher in diabetic than in normal group rats. Treatment withBBR (100,200mg/kg) and XKW (800mg/kg) significantly decreased FBG comparedwith untreated diabetic groups from the end of the6th week to the end of8th week. Thehigh-fat diet increased BW compared with the NC diet throughout the treatment period.Diabetic rats had lower body weights than HFC rats beginning at week2. Diabetic ratshad lower body weights than the NC rats beginning at week3. BBR treatment improvedBW, but the differences between these groups did not reach statistical significanceduring the entire study and remained lower than HFC rats.2Effects of BBR on biochemical and renal functional parameters in DN ratsThe U.A/C, UP/C, BUN, SCr, TG, TC and LDL-c levels of HFDC group weresignificantly higher, while HDL-c was significantly lower than those of the NC andHFC rats. This result indicates that our model was successful at inducing DN. When DNrats were treated for8weeks, BBR (100,200mg/kg) and Simvastatin (2mg/kg)significantly decrease the levels of U.A/C, UP/C, BUN, SCr, TG, TC, LDL-c andincrease the HDL-c level. Data indicate that BBR treatment could effectively improverenal function parameters and lipid metabolism in DN rats3Effect of BBR on renal histopathology of DN ratsCompared with NC group, the renal histology from untreated DN rats showed signs ofsevere pathological changes such as mesangial expansion, glomerular hypertrophy,inflammatory cell infiltration of the renal tubule-interstitium, the lumen was narrowed and part of glomeruli was sclerosis and there was a large number of collagen fibers inrenal tissue. In DN rats given BBR, the degree of pathological changes was significantlyreduced: thickening of GBM and mesangial matrix accumulation were scarcely detected,and inflammatory cell infiltration was also extremely reduced, although glomerularhypertrophy was observed to a small extent, and the expression of collagen fibers inrenal tissue was significantly decreased and the domain of interstitial fibrosis waslessened.4Effect of BBR on renal ultrastructureThe electron micrograph shows a normal ultrastructure of the glomeruli in NC. Inthe DN model rats, the glomeruli appears larger and dilated, the GBM was wrinkled andthickened, local fusion of the foot processes of the podocytes and podocytes loss wereobserved compared with NC group. Oral administration of BBR (100,200mg/kg) couldnormalize the above mentioned alterations to some extent.5Immunohistochemical stains for type IV collagen, FN, TGF-β1and CTGF inkidneyImmunohistochemical analysis of kidney tissue sections revealed that the expressionlevel of type IV collagen, FN, TGF-β1and CTGF, as brown or yellow granulae inglomeruli and tubules significantly increased in HFDC group compared with NC group,suggesting positive results. BBR (50,100,200mg/kg) treatment prevented theenhanced expression level of type IV collagen, FN, TGF-β1and CTGF on differentlevels.6Effects of BBR on the production of GRK2, GRK3, GRK4, GRK5and GRK6inrenal cortexGRK2, GRK3, GRK4, GRK5and GRK6, which are expressed in kidney, were analyzedby western blotting. The protein expression levels of GRK2and GRK3were enhancedapparently, while the expression of GRK4and GRK6were reduced sharply in renalcortex of DN model group compared to controls. Treatment with BBR (100,200mg/kg) not only down-regulate the protein expression of GRK2and GRK3, but alsoup-regulation protein expression of GRK6, GRK4level was slightly increased afterBBR treatment, but this difference did not reach statistical significance. There is nodifference on level of GRK5among these groups.7Effects of BBR on cAMP levelThe expression of cAMP in renal cortex of DN model group was much lower than thatof NC. BBR(100,200mg/kg)significantly enhanced cAMP level compared with DNmodel group. However, there was still a significant difference between them and that ofNC group.CONCLUSIONS1. The characteristics of therapeutic effects of BBR on DN rats mainly shows in thatBBR could improve renal function parameters, and improved FBG significantlyafter a long-term treatment, but has no obvious effect on the weight of DN rats.2. BBR effectively improved serum biochemical parameters and renal functionparameters in DN rats, suppressed the enhanced expression of type IV collagen, FN,TGF-β1and CTGF, and therefore relieved the renal injury of DN rats. The resultssuggested that BBR may exert renoprotective effects on DN rats.3. BBR markedly modulated the abnormality expression of GRKs protein, andelevated the decreased expression level of cAMP from renal cortex in DN rats,which indicated that G protein-AC-cAMP signaling pathway might be related to therenoprotection of BBR in high-fat diet and STZ-induced DN rats. |
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