Innovative Bioengineering Approach for Automated Analysis of Endoplasmic Reticulum Stress in Cochlear and Renal Tissues of Pharmacologically Treated Diabetic Rats

In recent decades, improvements in socio-health conditions and changes in dietary habits have contributed to a significant increase in the incidence of metabolic diseases, such as type 2 diabetes mellitus (T2DM) and obesity. The onset of these conditions leads to chronic metabolic alterations, frequently associated with systemic complications, including hypertension, vascular damage, and dysregulated glycemia, such as hyperglycemia. In addition to these well-known complications, less commonly recognized conditions, such as hearing loss, have been identified. Epidemiological studies suggest a correlation between diabetes and sensorineural hearing loss, although the mechanisms regulating this process remain unclear. In this context, endoplasmic reticulum stress (ERS), induced by hyperglycemia and hyperlipidemia, appears to be a potential cause of: a) damage to cochlear cells, contributing to irreversible auditory dysfunction and b) to renal damage, where diabetes is often associated with the development of diabetic nephropathy, one of the most severe renal complications, which is exacerbated by the activation of the renin-angiotensin system (RAS). A crucial role in ERS is played by the protein GRP78 (also known as Glucose-Regulated Protein 78). This molecular chaperone regulates endoplasmic reticulum stress by activating the unfolded protein response (UPR), which in turn regulates protein folding within the endoplasmic reticulum. There is evidence that it interacts with co-ligands to activate signaling pathways that promote cell proliferation and survival. For this reason, it is considered a potential therapeutic target. Losartan, a member of the ARB (angiotensin receptor blocker) medication class, primarily developed as an antihypertensive but with associated anti-inflammatory and anti-fibrotic properties, appears to show a protective function against hearing loss progression. Losartan appears to modulate GRP78 levels, potentially reducing endoplasmic reticulum stress, inflammation, and cellular damage. In this study, conducted at the Bioacoustics Laboratory of the Department of Neurosciences at the University of Padua, the Zucker Diabetic Fatty (ZDF) rat, a model characterized by obesity and type II diabetes, was employed to investigate the therapeutic efficacy of pharmacological treatment with Losartan in reducing endoplasmic reticulum stress and damage associated with the two target organs, ear and kidney. Histological and immunohistochemical analyses, performed on cochlear and renal sections, were supported by a customized algorithm developed in MATLAB for the automated processing of microscopic images. The parameters analyzed included intensity of immunoreactivity for GRP78 expression, nuclear counting in spiral ganglia, and morphometric assessment of the stria vascularis in cochlear sections. In renal samples, the intensity of immunoreactivity for GRP78 expression was quantified separately in the cortical and medullary regions. The results obtained demonstrated the efficacy of the pharmacological treatment in reducing inflammatory responses and cellular stress, suggesting a potential protective effect of the drug in treating the metabolic complications of diabetes, including its effect on cochlear tissues. Furthermore, a statistical analysis was performed to compare the placebo group with the Losartan-treated group and the lean sample, identifying any significant differences in protection from cochlear and renal damage.