Study Of Urinary Exosomal Phospholipase A2 Receptor In Diagnosis Of Membranous Nephropathy

Background: The discovery of phospholipase A2 receptor antibody and its antibody(a PLA2Rab)has opened a new era in diagnosing PLA2R-associated membranous nephropathy(PLA2R-MN)with a high specificity of 98%.However,the sensitivity was only 40% to 80%,and the cut-off value of a PLA2 Rab remains controversial.Exosomes,considered as a new kind of "liquid biopsy," have made a remarkable progress in diagnosis of many diseases.However,the role of exosome PLA2 R antigen in the diagnosis of PLA2R-associated membranous nephropathy has not been studied.Here we provided a preliminary report on this issue.Methods: The urine samples of 85 PLA2R-MN patients,20 healthy controls,and nephrotic controls(9 patients with SMN,21 patients with podocyte lesions and 4 PLA2 Rnegative MN patients)were collected and centrifugated for exosomes.Purified exosomes were identified by transmission electron microscopy,nanoparticle tracking analysis and exosome marker.Protein mass spectrometry was used for authenticating the specific peptides of urinary exosomal PLA2 R.Western blot served as a semi-quantitative method for assessing the exosomal PLA2 R level from all the urine samples.We then evaluated the efficacy of urinary exosomal PLA2 R antigen alone or combining with serum a PLA2 Rab level to diagnose PLA2 RMN.Moreover,the expression difference of urinary exosomal PLA2 R between PLA2R-MN and other groups was further verified by quantitative mass spectrometry.Results: Urinary exosomes contained a high level of PLA2 R antigen from 85 PLA2 RMN patients vs the nephrotic controls with other causes and 20 healthy controls,and the results of quantitative LC-MS also indicated that the average intensity of the PLA2R-MN group was to be at 9 orders of magnitude,which was 100-1000 times higher than that of the healthy group and the PLA2R-negative MN group or the SMN group and the podocyte injury group,respectively.Of note,the level of urinary exosomal PLA2 R antigen also had a good consistency with the PLA2 R antigen staining intensity in the renal specimens of these patients.The sensitivity of urinary exosomal PLA2 R for diagnosing PLA2R-MN reached 95.4%,whereas the specificity was 59.3%.Combined analysis of serum a PLA2 Rab and urinary exosomal PLA2 R detection showed that the proportion of patients with serum a PLA2 Rab values greater than 20RU/ml,between 2-20RU/ml and less than 2 RUml were 59.0%(36/61),13.1%(12/61),and 19.7%(8/61)respectively.Among 61 PLA2R-MN patients,only 5 patients(8.2%)were negative for urinary exosomal PLA2 R.Of them,1 patient had serum a PLA2 Rab value greater than 20 RU/ml(1.6%)and 4 patients had serum a PLA2 Rab value between 2 and 20 RU/ml(16.6%).The serum a PLA2 Rab values of patients with negative urinary exosomal PLA2 R were all less than 2 RU/ml.Combining detection of the urinary exosomal PLA2 R and serum a PLA2 Rab could develop a more sensitive diagnostic method for PLA2R-MN,especially for patients with serum a PLA2 Rab less than 2 RU/ml.We also found that urinary exosomal PLA2 R could be a valuable predictor of the treatment response.Conclusion: Urinary exosomal PLA2 R could serve as a sensitive biomarker for diagnosis of PLA2R-MN.