The Role And Mechanism Of Six1 In Acute Aristolochic Acid Nephropathy

Aristolochic acid nephropathy(AAN)is an acute and chronic evolutionary process.In acute aristolochic acid nephropathy,aristolochic acid mainly damages proximal tubular epithelial cells(PTEC),and apoptosis of renal tubular epithelial cells constitutes the major cell death pathway in the pathogenesis of acute AAN.Previous reports have confirmed that p53 pathway is involved in aristolochic acid-induced apoptosis of renal tubular epithelial cells.The homeobox gene Six1 is a transcriptional regulator that plays a role in cell development,proliferation,differentiation,and apoptosis.It also regulates the cell cycle and plays an important role in cell growth and proliferation.It has been reported that Six1 negatively regulates p53 and does not depend on p53 ubiquitination to degrade protein MDM2.If Six1 is overexpressed in the acute AAN model,it can reduce renal tubular epithelial cell apoptosis by regulating p53.Therefore,the purpose of this study is to investigate the protective role of Six1 in the regulation of acute aristolochic acid nephropathy by regulating p53 pathway and related mechanisms.Firstly,we established an animal model of acute aristolochic acid nephropathy by intraperitoneal injection of aristolochic acid(AAI),and found that apoptosis of renal tubular epithelial cells increased,and the expression of Six1 in the kidneys decreased,and the expression of p53,p-p53 and apoptosis-related protein cleaved-caspase3(c-caspase3)increased.We then used the renal subcapsular injection of adeno-associated virus(AAV9)to construct a mouse model of Six1 overexpression in the kidney.After the model was successfully established,a mouse model of acute aristolochic acid nephropathy was established on this basis.We observed that compared with the control group,the expression of Six1 increased,the expression of p53 and c-caspase3 decreased,the corresponding renal cell apoptosis decreased,and the tissue damage was reduced.Then we used a certain concentration of aristolochic acid to stimulate rat renal tubular epithelial cells(NRK-52E)in vitro,apoptosis increased,renal tubular epithelial cells Six1 expression decreased,while the expression of p53,p-p53 and c-caspase3 increased.Using adenovirus overexpression technology to overexpress Six1 gene in renal tubular epithelial cells,aristolochic acid stimulated the reduction of p53,p-p53 and c-caspase3 expression in renal tubular epithelial cells and apoptosis decreased.In summary,Six1 can down-regulate p53,thereby inhibiting apoptosis-related pathways,and ultimately play a role in reducing aristolochic acid-induced apoptosis of renal tubular epithelial cells.Therefore,increasing the expression level of Six1 is expected to become a new strategy for clinical treatment of acute aristolochic acid nephropathy.Part ? Expression of Six1 in acute aristolochic acid nephropathy Objective: To investigate the expression of Six1 gene in acute aristolochic acid nephropathy.Methods : The Nephromine(https://www.nephroseq.org)database of renal gene expressions was first searched for the expression of Six1 in human kidney disease.Then,the animal model of acute aristolochic acid nephropathy was established by intraperitoneal injection of aristolochic acid.The experimental animals were divided into two groups: 1.sham operation group(sham): saline injection;2.model group(AAN): aristolochic acid injection,AAI(20mg/kg)was injected intraperitoneally to induce acute AAN.The mice were sacrificed on day 1,3,and 7 after AAI injection.Serum levels of creatinine,urea,and other renal function were measured.PAS staining was used to observe the degree of tubule injury,and the expression and localization of Six1 was assessed by immunohistochemical staining.TUNEL staining was used to observe the apoptosis of renal tubular epithelial cells.The expression of Six1,p53,p-p53 and c-caspase-3 in kidneys was detected by Western blot and real-time PCR.Results:The expression of Six1 in kidneys of patients with CKD decreased significantly.In the acute AAN model,compared with the sham group,the expression of Six1 in the renal tubules was significantly decreased,and the apoptosis of renal tubular epithelial cells was increased.At the same time,p53,p-p53 and apoptosis-related protein c-caspase3(cleaved-caspase3)in the kidney.The expression of Six1 was negatively correlated with the expression of p53,p-p53 and apoptosis-associated protein c-caspase3.Conclusion:In the acute aristolochic acid nephropathy model,the decreased expression of Six1 was associated with increased apoptosis of renal tubular epithelial cells and increased renal pathological lesions.Part ? Six1 alleviates kidney injury by inhibiting p53-mediated PTEC apoptosis in acute AAN Objective:To investigate the protective effect and mechanism of Six1 in acute aristolochic acid nephropathy.Methods : The model of acute aristolochic acid nephropathy was established by intraperitoneal injection of aristolochic acid.The Six1 overexpression mouse kidney model was established by local injection of Six1 overexpressing adeno-associated virus(AAV9)under renal capsule.Two groups: 1 no-load virus+AAN model group(Vehicle+AAI): subrenal injection of no-load virus + aristolochic acid intraperitoneal injection;2 kidney subcapsular injection of Six1 over-expression virus + aristolochic acid intraperitoneal injection.21 days after the virus infection,AAI(20 mg/kg)was intraperitoneally injected to induce acute AAN.The mice were sacrificed on day 1,3,and 7 after AAI injection.Serum levels of creatinine,urea,and other renal functions were measured.PAS staining was performed to observe the injury degree of tubular,TUNEL staining was used to detect the apoptosis of renal tubular epithelial cells.The level of Six1 m RNA was detected by Real-time PCR.The expression of Six1,p53,p-p53 and c-caspase-3 protein in renal tissues was detected by Western blot.Results: In the acute AAN animal model,renal tubular epithelial cell apoptosis and renal pathological lesions reduced in the Six1 overexpression group.Compared with the empty group,the expression p53,p-p53 and apoptosis-associated protein c-caspase-3 decreased,but there was no statistical difference in renal function between the two groups.Conclusion:In the acute aristolochic acid nephropathy model,overexpression of Six1 gene can reduce renal pathological injury by down-regulating p53 and aristolochic acid-induced apoptosis of renal tubular epithelial cells.Part ? The Role and Mechanism of Six1 in Aristolochic Acid In Renal Tubular Epithelial Cell Injury Objective: To explore the role and mechanism of Six1 in aristolochic acid-induced renal tubular epithelial cell injury.Methods: In vitro,AAI was used to stimulate rat renal tubular epithelial cells(NRK-52E),and AAI concentration gradients and time gradients were set.Real-time PCR was used to detect the expression of Six1 m RNA.Western Blot was used to detect the expression of Six1,p53,p-P53 and c-caspase3 protein.Transfected NRK-52 E cells with adenovirus over-expressing Six1 gene were stimulated with AAI after transfection.Flow cytometry was used to detect apoptosis.Western blot was used to detect the expression of Six1,p53,p-p53,MDM2 and c-caspase-3.Results: With the increase of AAI concentration,the stimulation time prolonged,the expression of Six1 decreased,and the expression of p53 and p-p53 protein gradually increased in NRK-53 E.With the overexpression of Six1 gene,compared with that of no-load virus group.Apoptosis of AAI stimulated NRK-52 E cells was reduced,and the expression of p53,p-p53,c-caspase-3 and other proteins also decreased.There was no difference of MDM2 compared to the control group.Conclusion: Six1 can alleviate p53-mediated AAI-induced renal tubular epithelial cell injury.