| Background:Hypertension has become an important public health challenge worldwide due to its high incidence and risk of cardiovascular and renal diseases.In human hyperten-sion,proteinuria is a major sign of renal damage.Moreover,proteinuria is an importa-nt signals for activation that imflammatory cell infiltration in the tubulointerstitial and sustained fiber formation,which cause progressive renal damage.Chronic severe hypertension develops proteinuria is related to glomerular damage which mainly due to long-term high glomerular capillary pressure resulting in the dysfunction of the glomerular filtration barrier.The glomerular filtration barriers include podocytes,a fenestrated glomerular capillary endothelial system and intervening glomerular basement membrane(GBM).Hypertension is well known to affect the integrity of the podocytes,and podocyte dysfunction plays an essential role in the pathogenesis of proteinuria and glomerulosclerosis.Metformin has been widely used for the treatment of type-2 diabetes with minimal side-effects.The United Kingdom Prospective Diabetes Study(UKPDS)showed that metformin could reduce the incidence rates of macrovascular and microvascular disease.The possible mechanisms include reducing insulin resistance and inflammatory cells infiltration,improving endothelial function and fibrinolysis,influenc ing carbohydrate and lipid metabolism,and its anti-atherogenic and antiinflammatory effects.There are many studies demonstrated that metformin has a nephroprotective effect against diabetic renal damage through activates adenosine monophosphate-activated protein kinase(AMPK)signaling.Metformin significantly decreased the urine albumin excretion rate in patients with type 2 diabetes.Neverthel-ess,the exact mechanism of the renoprotective action of metformin remains unclear.Therefore,we have examined the effect of metformin on proteinuria and explore the mechanism in a model of the spontaneously hypertensive rat.Vascular endothelial growth factor(VEGF)-A,a proangiogenic growth factor highly expressed in the kidney,is critical for the establishment and maintenance of glomerular filtration.Vandetanib is a VEGF receptor inhibitor.Therefore,we mainly investigated the effect of metformin on VEGF secretion and proteinuria in rat kidney,and the effect of VEGF receptor inhibitor on proteinuria of 48-week-old spontaneously hypertensive rats(SHR)treated with metformin.Objectives:1.Does long-term metformin treatment reduce proteinuria and protect renal function in spontaneously hypertensive rats.2.To study the molecular mechanism of metformin reducing proteinuria.Methods:Animals experiment: A total of 59 male rats(age,8weeks)were used for this study.Of these,32 were spontaneously hypertensive rats(SHR)and 27 were control,Wistar-Kyoto rats(WKY).Both were obtained from Beijing Vital River Laboratory Animal Technology Co(Beijing,China).Twenty two SHR rats and nineteen WKY rats received metformin at a dose of 100 mg/kg per day oral gavage(SHR-Met rats and WKY-Met rats).Metformin was dissolved in the drinking water.The remaining 10 SHR rats and 8 WKY rats received drinking water alone and were used as metformin controls(SHR-Vehicle rats and WKY-Vehicle rats).All animals were inspected daily and weighed weekly.The treatment period lasted 10 months.Fasting blood glucose tested at weekly intervals.The foods were removed from animal cages for 12 h before the administration of fasting plasma glucose.Blood samples were collected at the end of tail vein.SBP was measured with the tail cuff method without warming the animals.An average SBP reading was taken from at least three consecutive recordings over 10-minute period.For estimation of urine protein,rats were individually housed in metabolic cages for 24 hours with free access to tap water and standard diet,and 24-hour urine was collected and the total urine volume was recorded.No rats died before each set of data was collected.At the end of the experiment,ten WKY rats and ten SHR rats treated with metformin were randomized to take place.Five WKY rats and five SHR rats received vandetanib at a dose of 12.5 mg/kg per day oral gavage(WKY-vandetanib group and SHR-vandetanib group).Vandetanib was dissolved in sorbitan 80.The remaining 5 SHR rats and 5 WKY rats received sorbose gavage 80 alone and were used as vandetanib controls(SHR-Vehicle group and WKY-Vehicle group).The treatment period is 2 weeks.To measure urinary protein,rats were housed individually in metabolic cages for 24 hours,given free drinking water and a standard diet,to avoid urine contamination,and 24-hour urine collection.No rats died before data collection.The remaining rats were anesthetized,kidney tissues were taken for histology and electron microscopic analysis,abdominal aorta blood was collected,and the supernatant was collected after centrifugation.Serum creatinine and blood urea nitrogen were measured.1.Rats were housed individually in metabolic cages for 24 hours;urine collect-ed for 24 hours was used to measure urinaty protein.2.Pathological analysis: The kidneys of rats were used for HE staining to observe the pathological changes of kidney in each group.3.Immunohistochemistry analysis: Immunohistochemistry was used to observe the expression of VEGF-A.4.Transmission electron microscopy: Transmission electron microscopy was used to observe the microscopic changes of glomerular filtration barrier.5.Enzyme-Linked Immunosorbent Assay: Enzyme-linked immunosorbent assay(ELISA)was used to measure the expression of VEGF-A in kidney tissue.6.Western blot: The expression of p-AMPK,AMPK-? and VEGF-A was detected by western blot.Results:1.Body weight,blood pressure,serum creatinine,blood urea nitrogen,fasting fasting blood glucose and serum insulin levels: There was no significant difference in body weight,serum urea nitrogen and fasting blood glucose in the final stages of the experiment(P>0.05).Metformin-treated rats had decreased serum creatinine.There was no significant difference in blood pressure between SHR rats and WKY rats,M-SHR rats and metformin-treated SHR rats.The serum insulin in SHR rats was higher than that in WKY rats in the control group.After treatment with metformin,serum insulin in SHR rats decreased(P<0.01).2.24-hour urine protein in rats: The urine protein of SHR rats was higher than that of WKY rats in the control group(P<0.01).After metformin treatment,the urine protein of SHR rats decreased(P<0.01);after vandetanib treatment,proteinuria of SHR rats already treatment with metformin of 48 weeks was significantly increased(P<0.01).3.Pathological analysis: HE staining showed increased infiltration of inflamm-atory cells(neutrophils,monocytes,and lymphocytes)in the tubulointerstitial area of SHR rats.Inflammatory cells in SHR rats were significantly increased compared with WKY rats.The number of inflammatory cells in SHR rats treated with metformin was significantly decreased(P<0.01).4.Immunohistochemical analysis: The expression of VEGF-A in kidneys of rats treated with metformin increased.5.Transmission electron microscopy analysis: Showed that the endothelial cells fenestrae in SHR rats was disappeared,and the endothelial cells fenestrae in SHR rats and WKY rats treated with metformin were obvious.SHR rats have extensive podocyte foot process fusion,but metformin-treated SHR rats and WKY rats have mild foot process fusion.6.Enzyme-linked immunosorbent assay: The expression of VEGF in kidneys of rats treated with metformin increased.7.Western blot: Western blot showed that the expression of p-AMPK and VEGF-A increased in the kidney of metformin-treated rats,while there was no difference in the expression of AMPK-? between the groups.Conclusion:Metformin reduces proteinuria in spontaneously hypertensive rats and has a protective effect on the kidney,which may be related to the involvement of upregulation of VEGF expression in the kidney. |
PDF Full Text Request