Establishment Of Nrf2-specific Knockout Mouse Model And Its Protective Role In Podocyte Indury

OBJECTIVE: Podocyte injury is the cause of most glomerular diseases,which leads to damage of the glomerular filtration barrier.The clinical manifestation is the massive loss of urinary protein,and the final progression to end stage renal disease(End stage renal Disease,ESRD).ESRD patients need to rely on renal replacement therapy to bring huge economic burden to patients and families.Most podocyte injury is caused by oxidative stress.Excessive reactive oxygen species(ROS)and insufficient antioxidant capacity are the main causes of tissue damage caused by oxidative stress.Oxidative stress is involved in the development of CKD(Chronic kidney disease).The Nrf2-ARE signaling pathway regulates the body’s redox homeostasis and is the most important defense mechanism against oxidative damage and drug toxicity.It is also involved in the regulation of cell cycle after stress and the regulation of inflammatory factors related to fibrosis.In view of the important regulatory role of Nrf2 in cell oxidative stress,autologous cell protection,cell cycle regulation after injury,and inflammatory response,we explored whether Nrf2 is involved in the development of podocyte injury and clarifies its development in glomerular disease.The role of this,in turn,provides new ideas and theoretical basis for the treatment of podocyte diseases in clinical work.Material methods and results:Part I: Expression and role of Nrf2 in podocyte injury1.Construction of podocytes specifically knocking out Nrf2 in miceThe Cre-Loxp system was used to construct the podocyte-specific PodocinCre/+/Nrf2loxp/loxp knockout mouse model,and the specific knockout model was successfully constructed by identifying the genotypes by rat tail DNA e×traction.At the same time,the immunofluorescence WT-1 and Nrf2 were used together.The localization method verifies that the specific knockout is successful.Results: There was no expression of Nrf2 in podocytes in podocyte-specific Nrf2 knockout mice.2.Pod-specific knockout of Nrf2 aggravates podocyte injury in mice with Adriamycin-induced nephropathyPodocinCre/+/Nrf2loxp/loxp and PodocinCre/-/Nrf2loxp/loxp male mice were randomly divided into FSGS(Focal sclerosing glomerulonephritis)animal models.The experimental group received a 10.5 mg/Kg dose of ADR in the penis,and the control group received an equal dose of Nacl in the penis to construct a doxorubicin nephropathy model.RESULTS: Podspecific knockout of Nrf2 aggravated the damage of adriamycin-induced nephropathy.After injection of ADR(doxorubicin),the PodocinCre/+/Nrf2loxp/loxp group died within 5 days,and the Podocin Cre/-/Nrf2loxp/loxp group had no death.The event happened.2.Observation of the natural course of podocyte injury in podocyte-specific knockout of Nrf2 miceWe explored the natural course of podocyte injury and podocyte-specific Nrf2 knockout.Urine was collected at week 7 until 29 weeks,and urine protein/creatinine ratio(UPCR)was measured.At 29 weeks,kidney tissue was collected,and part of it was used to extract protein and RNA for podocyte injury,cytokine,fibrosis and other related indicators.The other part was used for pathological section,PAS and immunofluorescence localization staining.RESULTS: There was a difference in urine protein between the 11 th weeks after the birth of podocyte-specific Knockout Nrf2 mice.The western-blot results showed that the e×pression of podocyte tumor-associated protein(Williams tumor-1,WT-1)was decreased.The results of PAS(Periodic AcidSchiff stain)showed that the podocytes of the mice were shed and the glomerular sclerosis was consistent with the performance of FSGS.The IF results showed that knockout podocyte Nrf2 significantly down-regulated the expression level of WT-1 in podocytes.3.Part II: CDDO-Me protect the ADR-induced nephritisIn this experiment,male BABC/L mice of 8-12 weeks were randomly divided into five groups and received overnight urine.Four groups were given CDDO-Me(Olean alkane)in each group 4 days before the start of the experiment.Triterpenoids)3mg/Kg or DMSO(dimethyl sulfoxide)was intraperitoneally injected every other day to stimulate systemic Nrf2.On the first day of the experiment,the FSGS model was established by intravenous injection of 10.5mg/Kg ADR or equal dose of Nacl into the penis.CDDO-Me 3mg/Kg or DMSO was injected,and the other group was given intravenous injection of 10.5mg/Kg ADR on the experimental day,and 100mg/Kg/d valsartan was administered as a positive control,which was collected after 5 days of treatment.Overnight urine,kidney tissue,part of paraffin section,pathological staining PAS,immunofluorescence,and another part of tissue protein extraction for molecular biological testing including WT-1,PODOCIN NEPHRIN.RESULTS: After 5 days of administration of the 10.5 mg/Kg dose of the ADR group,the urinary protein was significantly lower than the CDDO-Me group and was statistically different from the control group,which was the same as the positive control group.The results of IF(Immunofluorescence)indicated that the significant reduction of podocyte-associated protein levels in the CDDO-Me group was statistically different from the control group,and there was no difference between the control group and the positive control group.Conclusion: 1.Nrf2 has a protective effect on podocyte injury.2.The mechanism of action of Nrf2 in podocyte injury may be: affecting the expression of related proteins on podocytes and causing proteinuria,followed by podocyte shedding and fusion,and glomerular sclerosis.