Research On Glomerular Endothelial Cells Damage And Drug Protective Mechanisms In Pediatric Lupus Nephritis

Lupus nephritis(LN)is one of the most common complications that seriously affects the prognosis of systemic lupus erythematosus(SLE).LN is more common in children than adults.The early and long-term remission can effectively improve renal survival rate.At the initial stage,a large number of pathogenic autoantibodies and immune complexes in the serum contribute to oxidative stress in the entire endothelial system,including glomerular endothelial cell(GEC),which interacts with the immune response as the critical initiators in the development of LN.Glycocalyx,the first barrier of endothelial cells,is composed of proteoglycan and glycosaminoglycan.Syndecan-1(SDC-1),one of the proteoglycans,is connected to heparin sulfate(HS)in glycosaminoglycan to form the "skeleton" of glycocalyx.Glycocalyx shedding is associated with the oxidation reaction,leading to increased SDC-1 and HS in the blood and urine of patients.Nuclear factor erythroid-2-related factor 2(Nrf2)is a key transcription factor for oxidative stress,while heme oxygenase-1(HO-1)is an important antioxidant enzyme located in its downstream.Dl-3n-butyphthalide(NBP)can inhibit oxidative damage via Nrf2 activation.However,the mechanism of damage to GEC in LN children,the levels of SDC-1 and HS in blood and urine after glycocalyx injury,and potential mechanisms are poorly understood.Therefore,in this study,a series of experiments were conducted concerning the special pathological changes of GEC in LN children,the shedding of glycocalyx components,and mechanisms of protection and damage.Part One Clinicopathology and short-term prognosis of LN children with diffuse hyperplasia of GECObjective: To investigate the significance of diffuse hyperplasia of GEC in clinical manifestations,pathological features,early injury,induction therapy and therapeutic effect LN children.Methods: From March 2012 to October 2021,64 children with first-onset LN(type Ⅳ)initially diagnosed by renal biopsy and followed up at Hebei Children’s Hospital were enrolled,to collect the general information,laboratory reports,renal pathological results and short-term prognostic data(with a followup of at least 12 months or death as an endpoint).According to the degree of endothelial cell hyperplasia by renal biopsy,they were divided into diffuse hyperplasia of GEC group(group A),focal hyperplasia of GEC group(group B)and non-hyperplasia of GEC group(group C).The clinico-pathologic indexes of each group were analyzed statistically.According to remission at 6months after induction therapy,they were divided into com-plete remission group and incomplete remission group for univariate and multivariate analyses.Results: Among 64 children with LN(type Ⅳ),23(35.94%)children in group A experienced diffuse hyperplasia of GEC.Characteristics of the 23 patients included a short course of disease prior to hospitalization,high systolic blood pressure(SBP),increased systemic lupus erythematosus disease activity index(SLEDAI)score,low levels of complement C3 and C4,high levels of Ddimer(DD),low percentage of SSA and SSB,and increased urinary erythrocytes,leucocytes and urinary protein quantification,as well as reduced estimated glomerular filtration rate(e GFR)(P<0.05).The number of patients with glomerular microthrombosis on renal biopsy and basement membrane thickening under light microscope was increased,and the proportion of “onionskin” lesion was relatively low(P<0.05).Two children died during induction therapy,and the time to complete renal remission of the remaining patients was significantly shorter in group A than in the other two groups(P<0.05).Univariate analysis showed that factors affecting the complete remission at 6months after induction included diffuse hyperplasia of GEC,complement C3 and C4,DD,serum creatinine,urinary erythrocytes,urinary protein quantification,and glomerular microthrombosis(P<0.05).Diffuse hyperplasia of GEC and urinary protein quantification were statistically significant in multivariate analysis(P<0.05).Conclusions: The diffuse hyperplasia of GEC in LN children at the initial stage was different with respect to clinical manifestations,pathological features and remission after induction therapy,as compared with LN children with nondiffuse hyperplasia,which should warrant special attention as a relatively independent subtype.GEC may be involved in the pathogenesis of LN in children at early stage.Part Two Expression of markers of glycocalyx injury in blood and urine of children with LN and its potential mechanismObjective: To identify sensitive markers of glycocalyx shedding and to clarify the potential mechanism of damage to GEC by detecting the changes of SDC-1 and HS levels in blood and urine samples and the expressions of Nrf2 and HO-1 in GECs by renal biopsy in children with LN.Methods: Data were collected on 34 children with first-onset SLE that were initially treated in our hospital from October 2020 to October 2022 and were in remission at 6 months after induction therapy.They were divided into LN group and non-LN group according to presence of kidney involvement.Complete data of children that were qualified,including clinicopathological data,follow-up after treatment,blood and urine samples(before and at 6 months after treatment),were collected.Ten sex-and age-matched healthy children during the same period served as the control group.The levels of SDC-1 and HS in blood and urine samples were detected by enzyme linked immunosorbent assay(ELISA),and the differences between groups and before and after treatment were statistically analyzed.Pathological sections of 18 LN children undergoing renal biopsy were matched with those of 10 children with mild/minor lesions during the same period.The expressions of Nrf2 and HO-1in GECs were measured by immunohistochemical method,and the differences between groups were analyzed.Results: Before treatment,SDC-1 and HS in blood and urine samples showed no statistical difference between LN and non-LN groups(P>0.05),but significantly increased compared with the control group(P<0.05).After treatment,there was no significant difference in SDC-1 and HS in blood and urine between LN and non-LN groups(P>0.05).At 6 months after treatment,SDC-1 and HS in blood and urine of LN and non-LN groups were significantly lower than those before treatment,showing significant difference(P<0.05).The expressions of Nrf2 and HO-1 in GEC by renal pathology were significantly higher in LN group than in control group,with significant difference(P<0.05).Conclusions: The levels of SDC-1 and HS in the blood and urine of LN children were increased at the initial stage of the disease,but decreased with the remission of the disease.SDC-1 and HS from glycocalyx could be used as markers of disease activity and evaluation of therapeutic effect on LN.The damage to GEC in LN children may be related to the abnormal expression of Nrf2 and HO-1 in GEC.Part Three Mechanism of NBP protecting the glycocalyx by activating Nrf2Objective: To verify whether NBP could inhibit oxidation reaction by activating Nrf2,and stabilize SDC-1 and HS levels,thus reducing the damage to the glycocalyx and protecting cells by establishing in vitro cell models based on the effect of lipopolysaccharide(LPS)on HK-2 cells.Methods: The optimal LPS concentration,NBP concentration and Nrf2 siRNA sequences were screened.The experiments were divided into six groups:HK-2+siRNA NC group,HK-2+Nrf2 siRNA group,HK-2+siRNA NC+NBP group,HK-2+Nrf2 siRNA+NBP group,HK-2+LPS group and HK-2+LPS+NBP group.The apoptosis of cells in each group was detected by flow cytometry,and Nrf2 mRNA expression was detected by RT-PCR.Western blot(WB)was used to detect the expression of target protein Nrf2,E-cadherin,inducible nitric oxide synthase(iNOS),heparan sulfate proteog-lycan 2(HSPG2),SDC-1,thrombomodulin(TM),and tissue plasminogen activator(tPA).The differences between the groups were compared.Results: HK-2 cells were treated with LPS(10μg/ml)to establish in vitro cell model.100 μM was a nontoxic concentration for NBP,and Nrf2 siRNA1900 had the optimal knockdown effect.Transfection with Nrf2 siRNA significantly decreased the expression level of Nrf2 mRNA(P<0.05),reduced the expression levels of Nrf2,E-cadherin,HSPG2,SDC-1,TM and tPA protein(P<0.05),while significantly increased the expression level of iNOS(P<0.05).The addition of NBP led to increased expression level of Nrf2 mRNA and protein(P<0.05),enhanced expression levels of E-cadherin,HSPG2,SDC-1,TM and tPA protein(P<0.05),and significantly decreased iNOS level(P<0.05).Conclusions: NBP reduced iNOS protein in an in vitro model of HK-2cells by activating Nrf2,and increased the expression of Nrf2,E-cadherin,HSPG2,SDC-1,TM,and tPA protein,thereby protecting the glycocalyx and reducing cell apoptosis.