RAB27A silencing modulates miR-30d-5p expression and extracellular vesicle non-coding RNA cargo in diabetic podocytes

Diabetic nephropathy (DN) is one of the leading causes of end-stage renal disease and is characterised by early podocyte injury and loss. Increasing evidence indicates that extracellular vesicles (EVs) are key mediators of intercellular communication in the diabetic kidney, carrying bioactive molecules such as non-coding RNAs (ncRNAs) that influence disease progression. However, the identity of ncRNAs selectively loaded into podocyte EVs under hyperglycaemia and the contribution of vesicle-trafficking regulators such as RAB27A to this process remain to be elucidated. In this study, conditionally immortalised human podocytes were used to model diabetic conditions through high glucose (HG) exposure. To explore the role of vesicular trafficking in ncRNA release, RAB27A, an essential regulator of EV secretion, was silenced using siRNA. Small RNA sequencing was performed to characterize the EV ncRNA profile and subsequent RT-qPCR was used to validate results. Our findings revealed that HG and RAB27A silencing significantly altered the EV ncRNA composition, with a marked downregulation of miR-30d-5p. Additional experiments revealed that miR-30d-5p influences its target SMAD1, involved in antifibrotic signalling pathways. Collectively, these results propose that RAB27A-dependent vesicle trafficking and glucose stress regulate the ncRNA landscape of podocyte-derived EVs, positioning miR-30d-5p as a mediator of SMAD1-related pathways during hyperglycaemic stress.