Time-series proteomic study of the response of HK-2 cells to hyperglycemic, hypoxic diabetic-like milieu

Abstract

During diabetes, renal proximal tubular cells (PTC) are exposed to a combination of high glucose and hypoxic conditions, which plays a relevant role in the development of diabetic kidney disease (DKD). In this work, a time-series proteomic study was performed to analyse the effect of a diabetic-like microenvironment induced changes on HK-2 cells, a human cell line derived from normal proximal tubular epithelial cells. Cells simultaneously exposed to high glucose (25 mM) and hypoxia (1% O 2) were compared to cells in control conditions for up to 48 h. Diabetic conditions increased the percentage of death cells after 24 and 48 h, but no differences in the protein/cell ratio were found. The relative protein quantification using dimethyl-labeling and UHPLC-MS/MS analysis allowed the identification of 317, 296 and 259 proteins at 5, 24 and 48 h, respectively. The combination of statistical and time expression profile analyses indicated an increased expression of proteins involved in glycolysis, and a decrease of cytoskeletal-related proteins. The exposure of HK-2 cells to high glucose and hypoxia reproduces some of the effects of diabetes on PTC and, with the limitations inherent to in vitro studies, propose new mechanisms and targets to be considered in the management of DKD.

Comprehensive metabolomic study of the response of HK-2 cells to hyperglycemic hypoxic diabetic-like milieu

“…In total, 70.4 ± 5.51 µg of proteins could be obtained from the control and 60.2 ± 6.42 µg could be obtained from HG-hypoxia, which indicates a decrease of 15% in the protein content after the treatment ( p -value < 0.05 after two sample t test). These results are similar as those obtained in our previous work, and might correspond to the decrease of 15% on the cell viability observed after 48h 26 . It suggests that the experiment is being performed under similar conditions as before, indicating that the metabolite signals should be corrected by the protein content before any kind of comparison between the conditions studied.…”

“…To complement the metabolomics results, the 57 intracellular altered metabolites were combined with the 27 altered proteins (observed in our previous study 26 ) and analysed together using the Joint Pathway Analysis from MetaboAnalyst 4.0. This analysis provided a total of 4 significantly enriched pathways (Table 2 ), some of them already observed as enriched when using the Metabolite Set Enrichment Analysis.…”

“…In the present study, and to expand the knowledge on the DN research, a comprehensive metabolomic study has been performed to investigate the metabolic response of HK-2 cells to hyperglycemic, hypoxic diabetic-like milieu conditions. The experimental conditions selected were the same as in previous studies 25 , 26 , and the analysis of the extracellular medium was also performed. As it can be observed in the results section, the combination of different separation techniques (GC, HILIC and CSH, for lipidomics) coupled to high resolution MS (TOF and QE), the use of different ionization modes, and the application of advanced bioinformatics tools, freely available metabolite annotation tools, novel databases and libraries, and stringent cut-off filters allowed the annotation of more than 730 metabolites in the cell extracts and 290 metabolites in the extracellular media.…”

“…The combination of hyperglycaemia and hypoxia has been suggested to better mimic of the diabetic-like milieu environment, and the mechanism and consequences of the impaired Hif-1α response to these conditions have been described, highlighting the role of proteasome-dependent mechanisms of HIF-1α degradation 25 . More recently, a time-series study has been performed by our group to evaluate the response to HG-hypoxia at the proteome level, showing that short exposure times do not significantly affect the overall protein expression, but this expression is significantly affected when the treatment is sustained for 48h 26 . However, a comprehensive metabolomic study of the response of HK-2 cells to hyperglycemic, hypoxic diabetic-like milieu has never been performed.…”

“…The aim of the present study is to expand the knowledge of the DN progression through a comprehensive metabolomic study in cultured human PTC based on the combination of advanced analytical platforms (GC-time of flight (TOF) MS, HILIC-QExactive (QE) MS/MS and charged-surface hybrid chromatography (CSH)-QE MS/MS). The experimental conditions selected to study the intracellular metabolic changes were the same as in our previous study to complement the information obtained at the protein level 26 , but also including the analysis of the extracellular medium to expand the metabolic picture. Freely available metabolite annotation tools, novel databases and libraries, and stringent cut-off filters were applied to ascertain high quality data.…”