Advanced analytical methods for profiling disease-related alterations in diabetic patients with kidney impairment

In pursuing personalized therapeutic approaches, understanding molecular and biomolecular alterations in disease states is crucial for elucidating pathophysiological mechanisms and discovering predictive or diagnostic biomarkers. Investigations on complex samples eventually demand a combination of analytical approaches. The choice of the most appropriate and informative analytical approach depends on sample complexity and on the investigation’ aim/question, often requiring a multi-technique strategy for robust insights. In this PhD project, high-resolution mass spectrometry (HRMS) and surface plasmon resonance (SPR) were exploited to investigate structural and functional alterations in human serum albumin (HSA) within diseases characterized by enhanced oxidative stress and inflammatory response, with a specific focus on diabetic kidney disease (DKD). HRMS analyses in DKD patients revealed reduced levels of native albumin with concomitant increased levels of oxidized forms (mainly). The levels of both effective (eHA) and reduced HSA (rHA) forms dose-dependently correlated with renal dysfunction, showing higher diagnostic accuracy than total albumin (tHA) in DKD diagnosis. Moreover, eHA emerged as an independent predictor of DKD. To investigate whether structural alterations impact HSA binding properties, SPR technology was employed. Novel biosensing surfaces functionalized with a selective anti-HSA antibody were developed and validated for immunocapturing HSA directly from patients’ plasma to achieve a sensing surface snapshotting patient-specific HSA heterogeneity. Additionally, the biosensing surface facilitated the evaluation of novel bioresponsive prodrugs for tailored delivery systems targeting the tumor site specifically. Furthermore, the investigation delved into DKD-related metabolic changes using an untargeted metabolomic approach. The study highlighted progressive alterations in metabolic profiles, shedding light on potential key metabolites and pathways affected in DKD. In conclusion, the combination of HRMS and SPR technologies provided comprehensive insights into molecular changes associated with DKD. The final aim is to identify additional biomarkers for clinical use and deepen understanding of disease-related metabolic alterations, with potential applications for precise therapeutic interventions.