Proteomic analyses of Urine Exosomes reveal New Biomarkers of Diabetes in Pregnancy

Abstract

Objective To evaluate 24 hour urine exosome protein content changes among pregnant US subjects with diabetes and obesity during early pregnancy. Methods The exosome proteome content from 24 hour urine samples of pregnant subjects with gestational diabetes mellitus (GDM, N=8) and pre-gestational Type 2 diabetes (PGD, N = 10) were compared with control samples (CTRL, N = 10) obtained at week 20 of pregnancy. Differences in exosome protein load between groups was identified by liquid chromatography/mass spectrometry, analyzed by linear regression in negative binomial distribution, visualized in MetaboAnalyst (version 3.0), and validated by western immunoblotting. Results At the 20 th week of pregnancy, we identified 646, 734 and 856 proteins in exosomes from 24 hour urine samples of patients from the CTRL, GDM and PGD groups, respectively. S100 calcium binding protein A9, damage associated molecular pattern (DAMP) signal, was found to be significantly increased in both GDM and PGD subjects. In GDM subjects the peptide counts for S100A9 protein independently correlated with maternal obesity and macrosomia of the newborn infants. Early to late pregnancy developmental changes in the GDM group were shown to utilize pathways and protein expression levels differently from those in PGD or CTRL groups. Conclusions Urinary exosome proteomic analysis non-invasively provides insights into maternal changes during diabetic pregnancy. Exosome biomarkers early in pregnancy can be potentially used to better understand pathophysiologic mechanisms of diabetes at a cellular level, and to distinguish between gestational and pre-gestational diabetes at the pathway level. This information can aid intervention efforts to improve pregnancy outcomes in women with diabetes.

Exosomes as Messengers between Mother and Fetus in Pregnancy

“…Pathway analysis revealed that many of these proteins are involved in energy production and inflammation [104]. Analysis of urinary exosomes revealed that the damage associated molecular pattern (DAMP) protein S100A9 was present at higher levels in exosomes in GDM and could be used as valid biomarker of inflammatory processes and immune responses [105]. In addition, increased levels of miRNA members of the C19MC region were found in exosomes during GDM: miR-518a-5p, miR-518b, miR-518c, miR-518e, miR-520c-3p, and miR-525-5p [106,107].…”

Non-Coding RNAs and Extracellular Vehicles: Their Role in the Pathogenesis of Gestational Diabetes Mellitus

“…Bioinformatic analysis showed that the exosomal proteins in GDM target pathways were mainly associated with energy production, inflammation, and metabolism ( 113 ). In addition, the EVs proteome content from urine samples of pregnant patients with GDM was compared with that of controls, identifying 646 and 734 proteins in EVs from urine samples of controls and GDM patients, respectively ( 114 ). Notably, S100 calcium binding protein A9, a damage-associated molecular pattern signal, was found to be significantly increased in GDM ( 114 ), providing insights into maternal changes during diabetic pregnancy.…”

“…In addition, the EVs proteome content from urine samples of pregnant patients with GDM was compared with that of controls, identifying 646 and 734 proteins in EVs from urine samples of controls and GDM patients, respectively ( 114 ). Notably, S100 calcium binding protein A9, a damage-associated molecular pattern signal, was found to be significantly increased in GDM ( 114 ), providing insights into maternal changes during diabetic pregnancy. Further research to explore the role of types of proteins in EVs and clinical tests to confirm their importance are needed in the future.…”

A Critical Review of Proteomic Studies in Gestational Diabetes Mellitus

“…The concentration of circulating exosomes is found to be elevated in GDM cases [ 48 ]. Since circulating exosomes could be originated from the placenta and deliver to various types of cells, it is a novel and promising approach to elucidate the physiological mechanisms of GDM based on the proteins identified in the isolated plasma exosomes [ 41 ] or even urine exosomes [ 45 ]. It is generally accepted that the basic pathogenesis of GDM is insulin resistance.…”

“…First, peripheral blood and urine samples were drawn before diagnosis and used to identify biomarkers for early prediction [ 27 , 32 , 37 , 38 , 42 , 47 ]. Second, peripheral blood and urine exosome samples were also drawn at the same time with diagnosis and used to identify biomarkers for precision diagnosis or classification [ 29 , 31 , 41 , 45 ]. Third, placental tissues, umbilical blood, omental adipose tissues, abdominus skeletal muscle tissue, and colostrum whey were obtained during or after delivery and used to identify markers for the pathogenesis of GDM or fetal outcomes [ 28 , 30 , 33 – 36 , 39 , 40 , 43 , 44 , 46 ].…”