Hyperglycaemic Stress Due to Age/Rage Pathway Increases Dipeptidyl-Peptidase 4 and Extracellular Vesicle (EV) Release in Human Proximal Tubule Cells

Background: Diabetic kidney disease (DKD) is one of the major consequences of diabetes, tripling a patient's risk of death. DKD diagnosis requires urinalysis of albuminuria and kidney glomerular filtration rate (GFR), but these parameters are non-specific and late in identifying DKD. Thus, identifying novel markers for detection is crucial. The new indicators may be urinary extracellular vesicles (uEVs), released into urine by tubular and glomerular cells. We found that DPP4 (Dipeptidyl peptidase 4) was significantly associated with eGFR changes in a patient cohort with early DKD. We hypothesize that hyperglycemic stress promotes release of DPP4-containing EVs through the Receptor of AGE (RAGE) pathway and that DPP4-positive EVs can act as a diagnostic biomarker in the early detection of DKD. Methods: We induced hyperglycemic stress in HK-2 (human kidney) cells from the proximal tubule. Hyperglycemic conditions in cultured cells were mimicked by adding 50µg/ml of Advanced Glycosylation End Products (AGEs) to HK-2 cells. EVs were isolated from the cell conditioned media via differential centrifugation, generating low (P20) and high centrifugation pellets (P100). EV numbers and protein content (DPP4, RAGE) was monitored for changes using NTA (Nanoparticle tracking analysis) and immunoblotting. 50µM of TTP488 (Azeliragon) inhibited the receptor for AGE (RAGE)'s function. Results: AGE-treatment of HK-2 cells augments higher EV numbers release in different fractions (P20, P100) compared to controls (P100; Control: 3.1E9 particles/mL, AGE: 1.3E10; n = 40, p < 0.0001). Immunoblot DPP4 expression was also significantly higher in these EVs. However, inhibition of RAGE by TTP488 (Azeliragon) decreased EV numbers (P20; Control: 1.9E9/mL to 3.8E7/mL; P100; Control: 1.9E9/mL to 1.2E9/mL) and DPP4 protein content. Interestingly, RAGE was identified as an EV-specific cargo. Conclusion: Our studies support the hypothesis that DPP4 and MME positive EVs are released from tubular cells after stimulus with AGE. This aligns with our previous in-vivo data in DKD-patients. The DPP4/MME positive EVs, elevated in our in vitro and patient samples, could represent a novel tool to detect DKD earlier in disease-course than currently employed strategies. Funding: NIDDK Support, Private Foundation Support