To the best of our knowledge, this is the first study analyzing the differences of serum peptide profiles among healthy pregnant patients and those with T1DM. Using MALDI-TOF MS, and nano-MALDI-TOF/TOF MS, we have identified kininogen-1, complement C3, and C4-A as potential indicators for T1DM diagnosis in pregnancy. Consequently, validation with ELISA was utilized to quantify identified proteins. Kininogen-1 was found to be less abundant in sera of pregnant women with T1DM, while no significant difference in serum concentration was observed for complement C3 and C4-A in between the two groups.
Kininogen-1, also referred to as high molecular weight kininogen (HMWK), was identified as a peptide peak m/z 2081.79. It is believed to participate in the initiation of blood coagulation cascade, complement system activation, and is closely linked to the renin-angiotensin system via the angiotensin-converting enzyme (ACE). Kininogen is converted into a small peptide, kinin, by the enzyme called kallikrein. Kinins, specifically, are believed to be potent renal vasodilators with concomitant antithrombotic and antifibrotic functions. In the context of T1DM, kinins are believed to serve a protective role against the development of microalbuminuria and eventually diabetic nephropathy 15. An experimental study in mice has correlated kallikrein deficiency with the development of microalbuminuria. Similar results were observed with high ACE enzyme levels. ACE is known to convert angiotensin I into angiotensin II, however, it also plays a role of the kinin-degrading enzyme, resulting in significant degradation of kinin while having only a limited impact on angiotensin II production. Additionally, ACE I/D polymorphism has been previously associated with the development of diabetic nephropathy, while the ACE II genotype is believed to be nephroprotective in T1DM and T2DM 16. In fact, previous proteomic studies have associated differential expression of kininogen-1 with the evolution of microalbuminuria, thus allowing it to serve as an early marker of nephropathy associated with T1DM and T2DM 15, 17–19.
Vitova et al. 19 suggest an association between microalbuminuria in patients with T1DM and decreased activity of the kallikrein system. Specifically, they identified diminished urinary excretion of kininogen -1 heavy chain in T1DM non-pregnant patients with microalbuminuria compared to those without microalbuminuria. It was concluded that decreased activity of the kallikrein system, including kininogen, locally or systemically, was associated with the development of microalbuminuria. Since our study was meant to explore the potential serum biomarkers specific to T1DM in pregnancy, urine samples were not collected for proteomics. However, there were no women with diabetic nephropathy in our study group. If decreased systemic activity of the kallikrein system, is associated with development of diabetic nephropathy and associated complications, diminished serum kininogen-1 identified in our study may be an early marker of kidney function deterioration in pregnant women with T1DM not yet seen in routine urinalysis. Another study has also identified kininogen-1, as well as mannan-binding lectin serine protease 2, and prothrombin, as potential biomarkers for microalbuminuria in an attempt to prevent and diagnose diabetic nephropathy in T2DM patients at an early stage 17. Similar to the study by Vitova et al. 19 decreased urinary excretion of four identified biomarkers, including kininogen-1, was evident in patients with T2DM with microalbuminuria compared to those without microalbuminuria and healthy controls. Since all four identified biomarkers are believed to play a role in the complement cascade, decreased excretion was concluded to indicate dysfunction in immune response 17. Since both studies have evaluated urine proteomic profiles alone, while our study analyzed serum protein composition, the significance of the relationship between the two profiles, as well as the utility of either method for clinical evaluation of diabetic nephropathy, requires further examination. Controversially, a previous study in rats with T1DM have reported upregulation of kininogen levels in urine 20.
Another study utilized proteomic analysis of plasma samples to establish a correlation with early progressive renal function decline in macroalbuminuric patients with T1DM 21. Unlike in the current study, the mean abundance of kininogen-1 (three fragments) and a fragment of plasma kallikrein-sensitive glycoprotein (inter-alpha-trypsin inhibitor heavy chain H4, ITIH4) were increased by 30-50% in T1DM patients who were at risk of early progressive renal function decline, compared to those with normal renal function. Additionally, proteomic profiling in rats with induced T1DM revealed increased serum expression of kininogen in the aorta and the kidneys 22. Interestingly, this effect was believed to be modulated by hyperglycemia since treatment with insulin and control of blood glucose levels reversed the expression of kininogen. Although our study has identified decreased levels of kininogen-1 in serum, such discrepancy in result may be attributed to the animal model, level of blood glucose during the blood draw, or overall blood glucose control.
In the context of our result, decreased kininogen-1 in pregnant women T1DM may indicate a higher risk of developing diabetic nephropathy with microalbuminuria, which is associated with maternal and fetal complications. Diabetic nephropathy in pregnant women with T1DM was associated with a higher prevalence of preeclampsia (48%) and pre-term delivery (73%), compared to pregnant T1DM without diabetic nephropathy (preeclampsia – 24%, pre-term delivery 44%) 23. Also, intrauterine growth restriction was twice more common in pregnant women with T1DM and diabetic nephropathy compared to those with normal kidney function.
Interestingly, kininogen-1 (along with lumican) have been identified as potential biomarkers for late and early pre-term birth due to their differential expression in amniotic fluid samples 24. Wen et al. 25 have identified kininogen-1 as one of the 19 serum peptides that could serve as a predictor of preeclampsia (PE) or be used in the differential diagnosis of PE from confounding chronic hypertension 4,25. Our study group consisted of uncomplicated women who continued pregnancy up to term, however, it would be reasonable to design a new proteomic study in complicated diabetic pregnancies. If kininogen-1's utility as a biomarker is confirmed, it might be incorporated in routine screening in T1DM during pregnancy to assess the risk for development of diabetic nephropathy, associated complications including pre-term delivery and preeclampsia, or to develop early management strategies for such patients.
In the current study, complement C4-A and C3 were identified in serum based on fragments of m/z 1740.48; 1435.04; 1896.65 and 1865.35; 1519.27, respectively. However, unlike in the case of kininogen-1, there was no significant difference in their serum concentrations between the two study groups on validation using ELISA. The main aim of protein-peptide profiling is to compare the whole profiles established from MS spectrum data. Mathematical algorithms allow obtaining models based on the most characteristic features. However, the presence of a particular peptide in the created model does not always indicate that the whole protein would be dysregulated in the study group. Therefore, the additional validation of the obtained results is necessary. Classically, complement protein C3 is believed to play a role in the activation of the lecithin complement pathway, however, there is also evidence of its implication in insulin resistance. Studies of C3-deficient mice, however, have indicated decreased insulin level and improved glucose tolerance 26. Plasma levels of C3 mRNA in adipose tissue have also been negatively correlated with insulin sensitivity 27. Notably, serum complement C3 was shown to have a stronger association with insulin resistance than highly sensitive C-reactive protein in non-diabetic Chinese patients 28–30. In recent years, insulin resistance has been implicated in the pathogenesis of T1DM in pregnancy and may predispose patients to miscarriage, preeclampsia, and macrosomia 31. Downregulation of complement protein C3 was also observed in studies with T2DM patients 18,32,33. In studies of pregnant women with GDM, both maternal C3-A and C4-A concentrations were significantly lower compared to healthy women at the time of delivery 34. With that said, no significant difference in cord plasma levels of C3-A, C4-A and C5A was observed in women from both groups of the aforementioned study. Another study has noted a downregulation of C4-A in post-mortem testing of patients with sudden infant death syndrome 35.
On the contrary, analysis of vitreous humor in patients with diabetic nephropathy identified upregulation of complement protein C3 (along with apolipoprotein A1, APOH, fibrinogen, C4b, C9 and factor B) 36. Another study identified higher concentrations of glycated complement C4-A in patients with T1DM 37.
In the another study, amniotic fluid analysis of 15 women with preeclampsia and healthy controls did not reveal differences in C4A between the two groups 38. However, different study highlights the implication of C4A and apolipoprotein A-1 plasma level measurement in distinguishing women based on the onset and severity of preeclampsia 39. Significantly lower plasma concentrations of C4A were observed in women with severe, early-onset preeclampsia compared to those with severe, late-onset preeclampsia. Based on the current studies, there is no consensus about the roles of C4A and C3 in diabetes or pregnancy-associated complication. Based on the previous studies, the significance of C4 serum levels in T1DM during pregnancy remains unclear, and further investigations are required.
Identification of unique biomarkers in a setting of T1DM in pregnancy may be useful in early diagnosis and prediction of the risk of maternal and fetal complications as a result of disease progression. Overall, kininogen-1 has some utility in predicting microalbuminuria and diabetic nephropathy in patients with T1DM and T2DM, however, its utility as a biomarker in T1DM disease progression in pregnancy requires further clinical evaluation.
Proteome-wide profiling still remains to be a powerful tool in up-to-date science, however it is not free of limitations that have been addressed in previous projects 40,41. Our previous studies confirmed, that this approach is accurate for characterization and identification of proteomic patterns of different diseases 14,42. During the analysis, the m/z range of 1-10 kDa was examined, therefore, it could be assumed that only peptide fraction was analyzed. Peptide fraction in blood mainly occurs as the effect of natural proteolysis and depends on the activity and specificity of proteolytic enzymes, enzymatic stability of the particular peptide, and many others. Since, there is no certainty that obtained peptide pattern reflects protein composition, the immunoenzymatic tests (or other quantitative analysis) are necessary. However, the main aim of the profiling is to establish the specific fingerprint, characteristic for the study health condition, not an identification of a single marker. The additional identification of the differentiating m/z, may only suggest that the concentrations of the identified proteins are changed under the influence of the disease. The obtained results, which strongly suggest differences in the peptide composition, may reflect the occurrence of some changes in the process of proteolysis or in the proteins structures (see Table 4). Differential expression of proteases as well as protease inhibitors, has already been associated with other diseases, like breast cancer 43.