Insights into the role of the RAB3A-RAB27A system on the development of kidney injury associated with type II diabetes mellitus

Type 2 diabetes mellitus is an increasingly prevalent global health issue. It is well-recognised that type 2 diabetes mellitus is a leading factor contributing to renal damage, with up to 40% of patients progressing to diabetic nephropathy. Diabetic nephropathy is a common complication in diabetic patients with severe clinical implications, leading to end-stage renal disease. Kidney injury in diabetes entails, among others, damage in a key cell of the glomerular filtration barrier, the podocyte, which undergo loss and detachment from the basal membrane increasing the permeability of the glomerular filtration barrier. Communication between podocytes and other renal cells, mediated by extracellular vesicles, is vital for maintaining the intricate balance of renal function. This communication orchestrates the complex interplay of signalling cascades that contribute to the overall health and homeostasis of the kidneys. Within this framework, Rab GTPases play an indispensable role in regulating vesicle transport, which can impact an individual's health or contribute to disease onset. In a genetic association study, our team pinpointed a single nucleotide polymorphism in the RPH3A gene, an effector of Rab3A and Rab27A GTPases, to be associated with an increased risk for developing albuminuria. The central aim of this doctoral thesis was to investigate the role of the Rab3A-Rab27A GTPase system on the development of kidney injury associated with type II diabetes mellitus. Therefore, the components of this system were quantified in urine samples from patients with hypertension and diabetes and / or renal damage and the effect of elevated glucose and angiotensin II levels on human immortalised podocytes was evaluated. Moreover, this research delved into the silencing the Rab3A-Rab27A system by transient transfection through siRNAs in podocytes exposed to high glucose concentrations to assess its impact on podocytes physiology. To achieve these objectives, mRNA levels of podocyte markers and Rab GTPases were analysed by RT-qPCR, while protein levels were quantified through Western blot and confocal microscopy. Additionally, apoptosis, cytoskeletal arrangement, and vesicle distribution were assessed by immunofluorescence. Extracellular vesicles were characterised using several techniques, including nanoparticle tracking analysis, transmission electron microscopy, Western blot, flow cytometry, and super-resolution microscopy. Furthermore, microRNA expression levels in podocytes and podocyte-derived extracellular vesicles were quantified by quantitative RT-qPCR. The results showed that in urine pellets from hypertensive-diabetic patients, RAB3A and RAB27A mRNA levels were increased and showed a direct correlation with albuminuria. Rab3A and Rab27A protein levels were increased under glucose overload, and Rabphilin3A decreased. Furthermore, this system exhibited higher levels under stress conditions in a manner of angiotensin II dose and time treatment. Immunofluorescence imaging indicated different expression patterns of Rab27A under treatments. Silencing RAB3A and RAB27A leads to a decrease in podocyte differentiation markers and cytoskeleton organization, an increase in apoptosis and a CD63-positive vesicle cellular pattern distribution change. Interestingly, under high glucose, Rab3A-Rab27A silencing ameliorates some of these detrimental processes. Moreover, in both podocytes and extracellular vesicles derived from podocytes, we observed significant expression changes in miRNAs that regulate key pathways involved in diabetic nephropathy following silencing and glucose treatment. In summary, these findings suggest that the Rab3A-Rab27A system could be involved in the development of podocyte injury associated to glucose overload. Alterations of vesicular transport directed in part by this system, could be involved in a reduction in podocyte damage in diabetic nephropathy.