Systems biomedicine approach to diabetic nephropathy: role of coagulation protease-activated protein C
Previous studies suggested that the serine protease activated protein C (aPC) can prevent progression of diabetic nephropathy. In an animal model of diabetic nephropathy nephroprotection was associated with inhibition of glomerular apoptosis and mitochondrial dysfunction. The mechanism how the extracellular protease aPC prevents glomerular apoptosis and mitochondrial dysfunction remains unknown, but in other disease models aPC has been linked with altered gene-expression. Since recent studies suggested the involvement of microRNAs (miRNAs) in the development and progression of diabetic nephropathy, we performed microarray based mRNA and microRNA expression analysis of kidney samples from mice with and w/o diabetic nephropathy using wild type mice and two different mutants with single mutations affecting aPC availability (TMPro/Pro, low aPC plasma levels; and aPChigh, high aPC plasma levels). PCA (Principal Component Analysis) analysis of both types of microarrays and use of specific data analysis tools including the array-array correlation plot were employed to analyze the data. To draw conclusions about differentially expressed miRNA and mRNA a moderated t-statistics was used and a significance threshold of adjusted p value of 0.05.
MiRNA profiling revealed miR-34a-5 p as the only significantly upregulated miR in diabetic WT mice (as well as in both diabetic mutant mice) as compared to controls. We did not identify any differentially expressed miRs in direct comparison between both diabetic mutants, indicating that no direct post-transcriptional regulation with miR participation is likely to occur within aPC pathway. The analysis also demonstrated the highest number of differentially expressed miRs (n = 13) in diabetic TMPro/Pro vs. TMPro/Pro control mice. Seven of the detected miRs showed differential expression exclusively in this comparison: miR-1224 – 5 p, miR-2681, miR-494 – 3 p, miR-5105, miR-5109 and miR-5126, and miR-7e-5 p. This finding indicates that the deregulation of these miRs is associated with deactivation of the aPC pathway in diabetic nephropathy. MiR-26b-5 p was the only miR upregulated in diabetic aPChigh vs. diabetic WT mice, while miR-148a-3 p, miR199a-3 p, miR-31 – 5 p and miR-342 – 3 p were all found to be downregulated only in diabetic TMPro/Pro vs. diabetic WT.
We constructed two networks from genes de-regulated in diabetes in each mutant group, starting from DEGs from comparison between both diabetic mutants. Genes included in the networks interact directly with and are connected with DEGs from comparison of both mutants and should be considered as particularly important for mediating protection against diabetic nephropathy or it progression. Twelve of the aggravations signaling network genes are potentially regulated by four miRs, found specifically deregulated only in diabetic TMPro/Pro mice: let-7a-5 p, miR-494, miR-1224 – 5 p and miR-2861. These results warrant to be followed up in future studies.