| Chinese medicine holds that “the kidney governs the water”,the kidney qi governs the inspiration and regulates the metabolism of the whole body.In patients with kidney yang deficiency,renal transpiration,gasification,inducing astringency and promoting are impeded.Affecting the transport of body fluids,the main manifestations include soreness-tired of waist and knee,long urine and frequent nocturia.Kidney warming herbs can improve the above symptoms.Shenqiwan is the representative of Kidney warming herbs.The main treatment is soreness-tired of waist and knee,deficiency kidney dropsy,kidney-qi deficiency,etc.It is clinically used in patients with chronic kidney disease,such as chronic nephritis.In the course of the development of CKD,peritubular capillary damage generally occurs.Endothelial cell angiogenesis plays an important role in improving renal injury.Aquaporin is a family of cell transmembrane proteins that play an important role in maintaining fluid metabolism balance.After kidney injury,AQP1 promotes the migration of endothelial cells,promotes angiogenesis and vascular repair and is beneficial to the recovery of renal function.During the previous study,we found that the AQP1 levels were significantly reduced in rats with kidney-yang deficiency.Besides,Shenqiwan can improve the general symptoms of kidney-yang deficiency rats and increase the expression of AQP1.However,it is remains to be studied that whether Shenqiwan improve kidney damage by promoting angiogenesis of endothelial cells.Objective To observe the protective effect Shenqiwan on renal tubular capillaries in kidney-yang deficiency rats and the effect of human microvascular endothelial cells.To investigate whether Shenqiwan improve the rarefaction of peritubular capillaries is by regulating the expression of aquaporin 1 in human microvascular endothelial cells.Methods In vivo experiment Thirty male Sprague-Dawley rats were divided into five groups.Grouped as follows: the normal control group,the adenine group and the Shenqiwan group;six per group.Rats in the model group were given intragastrically 150 mg/kg adenine once daily for 35 days.Began from the 15 th,rats of Shenqiwan group was given Shenqiwan(1.5g/kg,3.0g/kg,6.0g/kg)intragastrically once a day for 21 days.The normal control group rats were given the same amount of normal saline every day.One hour after the last gavage,3% sodium pentobarbital solution was used for anesthesia.After blood was drawn from the heart,the heart,liver,spleen,lungs,kidneys and adrenal glands were rapidly separated.Take the supernatant of urine and blood.Serum ACTH,CORT,BUN,Scr,and urine 17-OHCS,U-TP levels were measured in each group.HE staining was used to observe the histopathological changes in the kidney;Immunohistochemistry was used to observe the distribution and expression of CD34,VEGF and AQP1 proteins in rat kidney;Contents of VCAM-1 and VEGF in renal tissues were tested by ELISA;The expression of AQP1 mRNA in renal tissue was detected by q PCR;The expression of MCP-1 and AQP1 protein in renal tissue was detected by Western blot.In vitro experiments Treatment of HMEC-1 cells with Shenqiwan containing serum at different concentrations(2.5%,5% and 10%).Microscopic observation,MTS and q PCR were used to observe the effect of Shenqiwan containing serum on cell morphology,cell proliferation and AQP1 protein expression.As the normal rat serum was control group,MTS method was used to detect the effect of containing serum on the proliferation of HMEC-1cells;Scratching method to observe the ability of cell migration;Tube formation assay to detected the ability of tube formation in HMEC-1cells;q PCR and Western blot methods were used to detect AQP1 mRNA or protein expression in HMEC-1 cells.AQP1 RNAi lentivirus was used to infect HMEC-1 cells.Then detect AQP1 mRNA level,protein expression,cell migration ability,tube formation ability with the intervention of the Shenqiwan containing serum.Results In vivo experiments 1.Compared with the normal control group,in the adenine group,serum BUN,urinary U-TP and Scr levels in the adenine group were significantly higher(p<0.01);Creatinine clearance was significantly reduced(p<0.01);The contents of ACTH,CORT and 17-OHCS decreased(p<0.01).The results of HE staining showed that the adenine group rats are kidney-yang deficiency rats,in which the internal structure of the glomerulus collapsed,the renal tubules dilated,the sediments were observed in the tubular lumen,and the renal tubules were obstructed.Compared with the model group,Shenqiwan at doses of 1.5g/kg,3.0g/kg and 6.0g/kg both improved renal pathological changes and significantly decreased the content of BUN,urine 24 h U-TP,and Scr(p<0.01);Increase the content of CRH,ACTH,CORT(p<0.01)and the content of 17-OHCS(p<0.01,p<0.01,p<0.05).And 3.0g/kg Shenqiwan can significantly increase creatinine clearance(p<0.01).2.Compared with the normal control group,the density of peritubular capillary in the adenine group was significantly decreased(p<0.01);VCAM-1 levels were significantly higher(p<0.01);VEGF levels were significantly lower(p<0.05);The expression of MCP-1 protein was significantly increased(p<0.01).It was shown that adenine-induced renal injury in rats has peritubular capillary damage.Compared with the model group,1.50g/kg,3.00g/kg,6.00g/kg Shenqiwan can significantly increase the levels of VEGF(p<0.05,p<0.05,p<0.01),decreased the levels of VCAM-1(p<0.01)and the expression of MCP-1 protein(p<0.01);3.00g/kg Shenqiwan can significantly increase the density of peritubular capillary in rats(p<0.01).3.Compared with the normal control group,the expression of AQP1 in the renal tissue of the adenine group was decreased(p<0.05,p<0.01,p<0.01).Shenqiwan with different doses can upregulate the expression of AQP1 mRNA and protein in different degrees.In vitro experiments 1.Compared with 10% normal rat serum group,5% Shenqiwan containing serum can maintain normal cell morphology,significantly promote proliferation of HMEC-1 cells(p<0.05,p<0.01)and increase the levels of AQP1 mRNA(p<0.01).Therefore,5% Shenqiwan containing serum was selected as the best concentration for follow-up experiments.2.Compared with the normal rat serum group,the cell migration rate and the tube formation ability of cells in Shenqiwan containing serum group was significantly increased.3.After AQP1 RNAi lentivirus infection of HMEC-1 cells,AQP1 mRNA levels and protein expression was significantly reduced;HMEC-1 cell migration,tube formation ability was significantly reduced;The ability of promoting cell’s migration and tube formation in HMEC-1 cells cultured with Shenqiwan containing serum was decreased significantly.Conclusion Shenqiwan can improve the peritubular capillary injury in kidney-yang deficiency rats.It can repair damaged blood vessels by upregulating the expression of AQP1 in human microvascular endothelial cells.It is suggested that Shenqiwan may protect the peritubular capillary damage in kidney-yang deficiency rats by up-regulating the expression of AQP1. |
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