Global proteomic analysis of insulin action in glomerular podocytes

Abstract

Introduction & Aim: Insulin signalling contributes to diverse cellular activities including protein synthesis, proliferation and cell survival. Insulin resistance describes the inability of cells to activate the insulin signalling pathway effectively. This leads to pathological effects in multiple organ systems including the kidney. In diabetic nephropathy (DN), there is progressive glomerular dysfunction and recent studies have demonstrated that the podocyte is a direct target for insulin action. Furthermore, abrogation of insulin signalling specifically in podocytes leads to progressive kidney damage and eventually kidney failure. This study utilised a global unbiased proteomic approach to examine insulin signalling in podocytes under normal and resistant conditions, with the aim of identifying key molecules that could be targeted for diagnostic or therapeutic purposes. Methods: A human immortalised, thermo-sensitive podocyte cell line over-expressing the human Insulin receptor (INSR) was used under normal or resistant conditions (induced by the free fatty acid palmitate). The INSR was isolated following insulin stimulation (100nM) from whole cell immunoprecipitation (IP) or plasma membrane using in situ immunoprecipitation (In situ-IP) at different time points, and label-free mass spectrometry (MS) was used to detect proteins interactions with the INSR in at the receptor level. Western blotting and immunofluorescene imaging (IF) were used for candidate validation and glucose uptake and the electrical cell-substrate impedance sensing assay (ECIS) was employed to assess functional responses to insulin stimulation. Results: The MS analysis of the INSR isolated from the human podocytes resulted in the detection of 26 known INSR direct binders, and the bioinformatic analysis also led to the identification of doublecortin domain-containing protein 2 (DCDC2) and the microtubule crosslinking factor 1 (MTCL1) as novel INSR binding proteins. The interaction of DCDC2 and MTCL1 with the INSR was confirmed by IP and IF, and under insulin resistance conditions DCDC2 displayed increased association with the INSR, while MTCL1 showed no significant change. The knockdown of DCDC2 and MTCL1 using specific siRNA in the podocytes resulted in abnormal cellular localisation of the INSR compared to controls, and the podocytes exhibited significant morphological changes. Furthermore, the knockdown negatively affected podocyte migration and cell-cell / cell-matrix junction formation. Conclusion: This study provides insight into the complexity and specificity of insulin signalling in podocytes, and may explain how insulin resistance can affect the integrity of the glomerular filtration barrier in chronic kidney disease such as DN. The bioinformatics analysis of the INSR complex led to the discovery and confirmation of DCDC2 and MTCL1 as novel INSR binding proteins, and the validation experiments demonstrated the involvement of these targets in insulin signalling and podocyte biology.

Date of Award	1 Aug 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Humphries (Supervisor) & Rachel Lennon (Supervisor)