Expression And Clinical Significance Of Urinary Exosomes And Exosomal Proteins Nrf2 And NLRP3 In Patients With Idiopathic Membranous Nephropathy

Objective:Idiopathic membranous nephropathy(IMN)has a large difference in prognosis.One third of patients can resolve spontaneously.Even one third of patients will eventually enter end-stage renal disease(ESRD)within 10-15 years.At present,the diagnosis and treatment of idiopathic membranous nephropathy are mainly based on urinary protein quantification,Estimated glomerular filtration rate(eGRF)and renal biopsy.Urinary protein quantification and eGFR may only reflect the results of kidney injury,but not the degree of renal pathological damage;renal tissue biopsy is an invasive test,and there are many puncture contraindications in clinical applications.Although the serum anti-Phospholipase A2 receptor(PLA2R)antibody level in IMN patients can be used as an important basis for the diagnosis of IMN,the evaluation of the disease,and the formulation of treatment plans,the positive rate of detection is only 50% to 72%.Therefore,It is essential for the treatment and management of IMN to find stable and non-invasive biomarkers.Exosomes are extracellular vesicles with a diameter of 30-150 nm,which encapsulate molecules such as lipids,proteins,RNA,and DNA.They are found in many body fluids,such as blood,urine,and cerebrospinal fluid.Under different conditions such as hypoxia,high sugar,and calcium oxalate,the exosomes released by the kidney's intrinsic cells into the urine and the signal molecules encapsulated by the exosomes will change.Therefore,urinary exosomes may be a good potential biomarker to reflect IMN renal tissue lesions.Methods:This study recruited 49 patients who were diagnosed with IMN by renal biopsy of Shengjing Hospital and 21 healthy volunteers with gender and age matching,and took 50 ml morning urine.And all patients must conform to the following inclusion criteria and exclusion criteria.None of the healthy volunteers had proteinuria,hypertension,diabetes,impaired renal function,or chronic inflammatory disease.Urine exosomes were extracted by differential centrifugation,and the diameter and morphological characteristics of exosomes were identified by transmission electron microscopy and particle size tracking analysis;Western blot analysis was used to detect exosomal marker protein ALG-2 interacting protein X(Alix)?Four-transmembrane protein 63(CD63)and tumor susceptibility gene 101protein(TSG101),successful identification of exosomes;we detected the expression of exosomal marker proteins(Alix,CD63 and TSG101)and exosomal proteins Nrf2 and NLRP3 by western Blot,and performed quantitative analysis;The relationship between the expression levels of exosomes and exosomal Nrf2 and NLRP3 in IMN patients and their clinical indicators(proteinuria and estimated glomerular filtration rate(GRF))or pathological changes of renal tissue(focal stage renal Glomerular sclerosis,renal interstitial fibrosis,complement C3 and PLA2 R immunofluorescence intensity)were statistically analyzed.The relationship between urinary exosomes and exosomes Nrf2 and NLRP3 expression and serum anti-PLA2 R antibody titers were analyzed.Results: Part I: the results of transmission electron microscopy,particle size tracking and Western Blot analysis showed that the urine exosomes can be successfully separated by differential centrifugation;the expression level of urinary exosomes in IMN patients was significantly higher than that of healthy controls,and proteinuria levels were positively correlated with renal tissue lesions(C3 complement and PLA2 R levels);ROC curves of urinary exosomes indicated that urinary exosomes can more easily distinguish patients with IMN from healthy controls;basic urinary extravasation during renal biopsy The expression level of exosomes in the effective group receiving conservative or immunosuppressive treatment was lower than the respective ineffective group;further statistical analysis found that urine exosomes and serum anti-PLA2 R antibodies were positively correlated.Part II: The expression levels of urinary exosomal proteins Nrf2 and NLRP3 in IMN patients were significantly higher than those in healthy controls,and were positively correlated with proteinuria levels,and were associated with renal tissue disease(segmental glomerulosclerosis,increased complement C3 deposition,and PLA2 R in renal tissue Increased antibody);ROC curves of urinary exosome proteins Nrf2 and NLRP3 indicated that urinary exosome proteins Nrf2 and NLRP3 can more easily distinguish patients with IMN from healthy control groups;basic urinary exosome protein Nrf2 during renal biopsy The expression levels of NLRP3 and NLRP3 in the effective group receiving conservative or immunosuppressive treatmentwere lower than those in the ineffective group;further analysis found that urine exosomal proteins Nrf2 and NLRP3 were positively correlated with serum anti-PLA2 R antibodies.Conclusions: 1.Urinary exosomes,exosomal proteins Nrf2 and NLRP3 were significantly increased in patients with IMN;2.Urinary exosomes,exosomal proteins Nrf2 and NLRP3 expression were related to proteinuria levels,and closely related to renal tissue PLA2 R,complement C3 fluorescence staining intensity,suggesting that it can reflect the degree of renal tissue activity and disease,can be used as an important indicator of IMN diagnosis and disease assessment;3.The expression levels of urinary exosomes,exosomal proteins Nrf2 and NLRP3 were correlated with serum anti-PLA2 R antibodies,and were closely related to the prognosis of patients,suggesting that they can be used as one of the prognostic indicators for IMN evaluation.