#1849 Wide-proteome analysis on renal tissue samples reveals new intriguing outcomes on NA-Milan hypertensive rat strain phenotype

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Abstract Background and Aims Hypertension (HT) is a complex and heterogeneous pathological condition: the interaction between genetic and environmental factors leads to an increase in blood pressure (BP). HT represents a risk factor for cardiovascular disease and the major cause of mortality in industrialized countries, affecting 20% of the adult population. One of the environmental factors that plays an important role in the development of HT is sodium intake. The regulation of renal sodium and chloride are fundamental for the management of BP levels. Sodium sensitivity is a crucial factor that determines a more complex patient phenotype. The link between BP regulation, HT, sodium sensitivity, and HT-mediated organ damage has not yet been fully explored and is currently an unmet clinical need. To date, the change of molecular signature in the kidney from normal to hypertensive state is not entirely clear. It is possible to investigate this endpoint by having a rat model which spontaneously develops HT. Hypertensive rats called NA are congenic and carry a missense mutation in the alpha-Adducin 1 (ADD1) gene associated with HT. These rats developed HT in the form of early increase of systolic blood pressure (SBP) and proteinuria however without changes in GFR. To deepen our knowledge on the multi-omic field the following study aims to compare these outcomes integrating the data with profiling of renal tissue proteome in normal and hypertensive state of rats. Method Rats of MN and NA strains, starting from 4 up to 16 weeks of life, were enrolled to the study. The MN rat strain is the normotensive one, representing the non-pathological control of NA rats. Phenotyping was conducted on male rats over the time in terms of SBP, controlled with tail cuff method, evaluations of kidney physiology (transdermal monitoring of GFR in awake rodents with MediBeacon device), oximetry, blood gas analysis and urinary excretion of proteins and electrolytes. A label-free quantitative proteomic analysis was performed on renal cortex samples, carefully collected and stored to ensure optimal downstream processing, using nano-LC–HRMS (nano liquid chromatography–high-resolution mass spectrometry), with a Dionex Ultimate 3000 nano-LC coupled to an Orbitrap Fusion Tribrid mass spectrometer. Data were processed with MaxQuant v.1.6.0, while Perseus v.1.6.1.43 facilitated statistical analysis. Differentially regulated proteins were analyzed to identify modulated pathways across experimental groups, with functional insights obtained using STRING v.1.7.0 and Ingenuity Pathway Analysis (IPA). Results Physiological evaluations conducted on rats revealed an SBP increase starting from 12 weeks in NA rats compared to MN relative controls. At 16 weeks of age SBP reached in hypertensive strain a mean value around 160 mmHg (P = 0.039) (Fig. 1a). The parameter of urinary ratio of total proteins to creatinine was higher in NA rats starting from 12 weeks and a significative variation was visible at 16 weeks (P = 0.014) (Fig. 1b). The same trend has been observed for urinary total protein excretion after 24 hours with a significative variation between the two strains starting from 12 weeks (P = 0.0029) (Fig. 1c). Proteomic analysis revealed a clear modulation of pathways associated with inflammation, heme-toxicity, ferroptosis and fatty acid metabolism. In NA rats, starting from week 8, distinct markers of oxidative phosphorylation dysfunction emerged, indicating mitochondrial alteration and a consequent oxidative stress state. Furthermore, amino acid metabolism pathways were profoundly dysregulated, along with sodium transport mechanisms, as shown in Fig. 2. Conclusion Our pre-clinical study developed in vivo confirmed the hypertensive phenotype of NA rats in terms of early increase of SBP and proteinuria. The intriguing outcomes from proteome analysis suggest early kidney damage secondary to hypertension and sodium retention which exacerbates the energetic metabolism. Further studies are ongoing to better investigate the role of HT-associated biomarkers of prognostic/diagnostic value integrating the obtained data with single cell RNA-sequencing profile.

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