MUC1/CD9 DOUBLE-POSITIVE URINARY EXTRACELLULAR VESICLES FOR NON-INVASIVE DIAGNOSIS OF CHRONIC KIDNEY DISEASE

Chronic Kidney Disease (CKD) is a global health priority, necessitating urgent development of non-invasive, high-accuracy diagnostic tools due to the large number of undiagnosed cases. Furthermore, calculating eGFR requires blood sampling in a hospital setting, which presents a barrier to widespread screening. Urinary extracellular vesicles (uEVs) carry proteins shed from renal cells and serve as promising novel biomarkers. We previously found that the molecular expression profile of uEVs is useful for screening pediatric CKD, and we developed a simple ELISA method to quantify the expression of several molecules, including MUC1 (iScience 2022, STAR Protocol 2023). We aimed to establish a high-throughput ELISA method for quantifying uEV-surface protein MUC1 and to evaluate its diagnostic accuracy for CKD in both pediatric and adult cohorts.

A high-throughput sandwich ELISA protocol was developed for uEV-MUC1 quantification, utilizing plates coated with either Tim-4 (recognizing phosphatidylserine) or an anti-CD9 antibody for uEV capture, and a sensitive anti-MUC1 monoclonal antibody for detection. The assay's performance was evaluated in 930 samples from 470 adults and 88 children (ranging in age from 0 to 91 years, with a median age of 37 years) with diverse CKD etiologies. Diagnostic accuracy of uEVs-MUC1 was assessed using ROC curve analysis for detecting decreased renal function (eGFR <60). Additionally, stability tests were conducted under various urine storage temperatures.

uEV-MUC1 levels showed a significant decrease corresponding to advancing CKD stage in both adult and pediatric cohorts. Both the raw uEV-MUC1 measurement and its uCre-normalized value (uEV-MUC1/uCre) showed high diagnostic accuracy for CKD (eGFR <60) in adult and child (AUC>0.87). The diagnostic accuracy of uEV-MUC1 was slightly higher with the CD9 capture plate than the Tim4 capture plate, and both methods showed significantly higher accuracy than urinary protein alone (AUC 0.75). When restricted to cases with glomerular disease (nephrotic syndrome, IgA nephropathy, or diabetic kidney disease) (AUC>0.90), or cases without urinary protein, uEV-MUC1 consistently reflects renal function regardless of underlying disease. For clinical feasibility, MUC1 measurements in urine stored at 4°C remained stable for at least two weeks, comparable to -80°C storage. Furthermore, highly reproducible results were obtained even with samples stored at -80°C for over one year.

The ELISA-based uEV-MUC1 assay offers a non-invasive, highly accurate biomarker for CKD diagnosis, reflecting renal function decline regardless of disease or age. Mechanistically, high accuracy is achieved by selecting CD9 and MUC1 double-positive uEVs. Considering that MUC1 is expressed across the Henle's loop, Distal Convoluted Tubule, and Collecting Duct (CD), while CD9 is expressed only in the CD, the high diagnostic capability of the MUC1 and CD9 combination suggests that the marker is specifically capturing quantitative or qualitative changes in the CD, which may be a common feature shared across various CKD etiologies. Combined with the marker's superior diagnostic performance, elimination of the need for blood sampling, and stability under refrigeration, this assay strongly supports its potential for integration into general CKD screening programs, thereby facilitating earlier detection and intervention.