Poster Presentation 25th Annual Lorne Proteomics Symposium 2020

Association of post-translational modifications in the evolution of diabetic nephropathy in a rodent model of type 2 diabetes mellitus (#111)

Shivanjali J. Lingam 1 , Desmond K. Li 1 , Stuart J. Cordwell 2 , Melanie Y. White 2
  1. Discipline of Pathology, School of Medical Sciences, Charles Perkins Centre, University of Sydney, Sydney, New South Wales, Australia
  2. Discipline of Pathology, School of Medical Sciences, School of Life and Environmental Sciences, Charles Perkins Centre, University of Sydney, Sydney, New South Wales, Australia

Type 2 diabetes mellitus (T2DM) mediated renal dysfunction (diabetic nephropathy (DN)) is a progressive chronic complication which further increases the risk of T2DM mortality. Several animal/human and tissue/cell-based studies have found reactive oxygen and nitrogen species (ROS/RNS) as key mediators of signal transduction during T2DM and DN leading to increased oxidative stress.  The increased capacity for this T2DM-induced oxidative stress modulates protein function by accelerating post-translational modifications (PTMs) of protein residues. Here, we examined the cysteine redox and phosphorylated-modified renal proteome in T2DM. Rats were fed a standard CHOW (C) (12% fat) or high fat (HFD) (42% fat) diet for 8 weeks, with T2DM induced in 50% of the animals after 4 weeks utilising a low dose of streptozotocin (STZ; 35mg/kg); a pancreatic β-cell toxin. The remaining 50% were injected with a buffer vehicle (Cit). At the cessation of the feeding protocol, 9 animals per treatment group (CHOW/Cit; CHOW/STZ; HFD/Cit; HFD/STZ) were euthanised and kidneys excised. Tissue lysates were used to perform histological analysis to confirm the presence of DN pathological features of glomerulosclerosis and hyalinisation more pronounced in the HFD/STZ group. Tissue lysates were also subjected to western blotting using anti-glutathione, anti-sulfenic acid, and anti-S-nitrosylation antibodies. Western blots revealed gross changes in glutathionylation, sulfenylation and nitrosylation PTMs associated with diet and pathology. To identify discrete sites targeted by phosphorylation and redox PTMs, we performed isobaric tagging (TMT) and enriched for modified peptides using thiol-affinity chromatography, with subsequent analysis by LC-MS/MS. Significantly modified proteins, changing in abundance between one or more treatment group in comparison to CHOW/Cit, were mapped to enriched regulatory pathways. Our data suggests that the rat renal redoxome and phosphoproteome are sensitive to diet and T2DM, indicating a possible physiological remodelling role of oxidation and phosphoproteome in DN.