| Background:Systemic Lupus Erythematosus(SLE)is an autoimmune disease characterized by overproduction of autoantibody,immune complex formation and deposition in different parts of the body,leading to multiple organ damage.Lupus nephritis(LN)is one of the most common and serious complication of SLE.In the first five years of diagnosis of SLE,50%-70% of patients develop lupus nephritis and may cause permanent kidney damage and chronic kidney disease.In LN patients,podocyte damage is common."Pure podocyte disease" may be a form of podocyte damage in lupus nephritis.The foot process width(FPW)of patients with IV~V LN was significantly different from the normal level,and FPW was associated with proteinuria(r = 0.509,P< 0.001).Inhibition of podocyte injury may become a new treatment for LN.Autophagy preserves the homeostasis of the body by lysosomal degradation of cytoplasmic contents.Based on the way cytoplasmic contents are delivered to lysosomes,scientists have identified three major types of autophagy: macroautophagy,microautophagy,and chaperone-mediated autophagy(CMA).Among them,macroautophagy is the best understood one.Macroautophagy(referred to as autophagy)describes the degradation process,forming unique bi-membrane vesicles(autophagosomes)to isolate a portion of the cytoplasm,which is then fused to lysosomes to form autolysosomes and degrade internal matter.Recent studies on vitamin D have shown that its role in disease is not only the regulation of traditional calcium and phosphorus,but in tumors,autoimmune diseases,and kidney diseases,vitamin D affects the progression of disease by regulating autophagic activity.Small cell lung cancer,Crohn's disease,chronic kidney disease,diabetic nephropathy and other diseases have been reported.The prevalence of vitamin D deficiency in SLE patients in Henan province,China,showed that the prevalence of vitamin D deficiency was as high as 97.52%,especially in 34.71% of patients with severe vitamin D deficiency.Petri et al.found that urine protein levels were reduced by 15% after serum-active vitamin D levels increased by 20 ng/ml.Previous studies have shown that vitamin D has a protective effect on podocyte injury.However,it is not known whether vitamin D exerts a podocyte protection effect on LN patients through an autophagic pathway.The aim of this study was to investigate whether vitamin D protects against podocyte injury induced by autoantibodies purified in the serum of patients with lupus nephritis(LN)by autophagy.Methods:In this study,kidney tissue and immortalized human podocytes were selected as research objects.1.Collection of human kidney tissue specimens: 25 cases of renal tissue specimens of LN patients were selected(type III n=5;type IV n=5;type V n=5;type III+V n=5;type IV+V n=5),7 cases of healthy kidney tissue other than 2cm adjacent to the tumor after renal tumor resection were taken as control.The specimens were taken from Department of Nephrology and Urology of the First Affiliated Hospital of Zhengzhou University.2.Collection of serum tissue from peripheral blood of patients: collect serum sample of patients diagnosed with lupus nephritis,and store serum after centrifugation for preparation of autoantibodies in serum.The specimens were taken from the First Affiliated Hospital of Zhengzhou University.3.Transmission electron microscopy(TEM)was used to observe the level of autophagy activity in renal tissue of patients with lupus nephritis(LN).Patients with lupus nephritis(LN)were observed under transmission electron microscope(type III n=5;type IV n=5;type V n=5;type III+V n=5;type IV+V n=5).The number of autophagosomes in podocytes,endothelial cells,mesangial cells,and renal tubular epithelial cells in kidney tissues was counted and statistically analyzed.4.Correlation analysis of 24-hour urine protein,renal pathological activity score and number of autophagosomes in patients with lupus nephritis(LN).5.Count the number of autophagosomes in LN patients and measure the serum active vitamin D concentration for correlation analysis.6.To determine the effect of IgG on human podocytes cultured in vitro and the extent of autophagy,and to observe the role of vitamin D in podocyte injury.The IgG(1.5 mg/ml)in the serum of the purified LN patients was applied to human podocytes cultured in vitro,and vitamin D(100 nmol/L)was added to observe changes in autophagy index and podocyte related indexes.The podocytes were observed under high power transmission electron microscope.The autophagy activity was evaluated by Western blot analysis and qRT-PCR.The mRFP-GFP-LC3 B adenovirus was transfected into human podocytes in vitro and observed under laser confocal microscopy.The number of yellow fluorescence was counted and statistically analyzed.Result:1.Significantly higher levels of autophagic activity were observed in renal tissue of LN patients(P < 0.05).It is worth noting that in the natural cells of the kidney,podocytes showed higher levels of autophagy(P < 0.05).Among the different types of LN,the autophagy levels of type V,III + V and IV + V were higher than those of type III and IV.2.The number of autophagosomes in LN patients was positively correlated with the 24-hour urine protein and negatively pathological index AI(P <0.05).3.The levels of autophagy in podocytes of patients with different vitamin D concentrations were different,and there was a correlation after statistical analysis(P<0.01).4.The expression of LC3B-II and Beclin 1 in serum-purified IgG-treated group of patients with lupus nephritis was observed a significant increase in vitro,and the expressions of p62 and nephrin were decreased.PCR results showed increased gene expressions of MAP1LC3 B,BECN1 and decreased expression of SQSTM1 gene,more autophagosomes were observed under transmission electron microscopy,and more LC3 B spots were observed under confocal microscopy in the IgG group,indicating induction of autophagy and podocyte injury.Decreased autophagic activity and reduced podocyte damage were observed in the IgG + VD group.Conclusion:1.The level of autophagic activity in renal tissue of patients with lupus nephritis is higher than that of healthy controls,and the level of autophagy in podocytes of type V,type III+V and type IV+V LN are more significant than type III and type IV.2.Serum purified IgG in patients with lupus nephritis can lead to podocyte injury and induce autophagic activity in vitro,vitamin D can reduce aberrant autophagy and thus alleviate podocyte injury.3.This study demonstrates that vitamin D plays a protective role in autoantibody-induced podocyte injury in LN patients,which may become a novel therapeutic target in LN. |
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