Identification and evaluation of novel biomarkers in diabetic kidney disease

Abstract

Introduction: Diabetic kidney disease (DKD) is a progressive microvascular complication that leads to a decline in renal function and is the most frequent cause of end-stage renal disease. Currently, there is a need for improved biomarkers for the early detection of DKD. microRNAs (miRNAs) are short, non-coding regulatory ribonucleic acid (RNA) molecules that are commonly found in bio fluids and may reflect differential gene expression ongoing in the kidney during the disease process. Emerging evidence has shown that systemic and local immune responses contribute to the pathophysiology of DKD. Lymphocyte activation gene 3 (LAG3) has been known for its role in immune reactions. Additionally, sequence differences within the gene such as singlenucleotide polymorphisms (SNPs) may alter how a gene is transcribed, or cause a change of amino acid and affect the function of the translated protein. Aims and objectives: The primary aim of this thesis was to identify novel biomarkers associated with renal dysfunction in diabetes and to identify novel genetic susceptibility related to it. The study aim was addressed through three individual studies detailed below. Methods: Participants were recruited to three different studies. The first study comprised discovery and validation cohorts, applying quantitative polymerase chain reaction (qPCR) based techniques to identify and quantify miRNA biomarkers associated with renal dysfunction. Eighty-seven human urinary exosomal miRNAs were profiled in the discovery cohort of patients with type 2 diabetic kidney disease (T2DKD), (n = 14) and age and gender matched controls with type 2 diabetes mellitus (T2DM) and normal renal function (T2DNRF; n = 15). Independent validation of differentially expressed miRNAs was performed in a second cohort with T2DKD (n = 22) and two control groups: T2DNRF (n = 15) and non-diabetic controls with chronic kidney disease (CCKD) (n = 18). The second study applied cutting-edge miRNA next generation sequencing (NGS) techniques to examine differential expression of miRNAs in plasma samples associated with T2DKD, followed by independent replication and validation of altered miRNA profiles. The discovery cohort contained individuals with T2DKD (n = 9), age and gender matched patients with T2DNRF (n = 13) and persons with non-diabetes mellitus and normal renal function (NDNRF, n = 11). Differentially expressed miRNAs from the discovery cohort were reassessed in a second cohort with T2DKD (n = 10) and two control groups: T2DNRF (n = 10) and NDNRF (n = 10). The third study was a case-control study using deoxyribonucleic acid (DNA) Sanger sequencing of exons of the LAG3 gene in white participants of European ancestry, to investigate the association of LAG3 gene SNPs with DKD. Forty-one T2DKD, 29 T2DNRF and 97 NDNRF controls were investigated. Results: In the discovery cohort of the first study, urinary miR-21-5p, let-7e-5p and miR-23b3p were significantly upregulated in T2DKD compared to T2DNRF (p < 0.05). Conversely, miR-30b-5p and miR-125b-5p expression was significantly lower in T2DKD (p < 0.05). Independent validation confirmed up-regulation of miR-21-5p in the replication cohort in T2DKD (2.13-fold, p = 0.006) and in CCKD (1.73-fold, p = 0.024). In contrast, miR-30b-5p was downregulated in T2DKD (0.82-fold, p = 0.006) and in CCKD (0.66-fold, p < 0.002). There was differential expression of two miRNAs between T2DKD and T2DNRF (corrected-p < 0.05) in the discovery cohort of the second study. The expression profiles of both miRNAs were significantly reduced in T2DKD; miR-144-3p (0.25-fold, p = 2.15 x 10-5 ) and miR-190a-5p (0.17-fold, p = 3.24 x 10-5 ). Independent validation confirmed reduced expression of miR-190a-5p in the replication cohort in T2DKD (0.53-fold, p = 0.002). In the third study, no LAG3 genotype was significantly associated with DKD. However, there was a significant difference in plasma LAG3 protein levels across T2DKD (912.2 ± 609.6 pg/ml), T2DNRF (514.9 ± 401.9 pg/ml) and NDNRF (908.0 ± 787.6 pg/ml). Additionally, plasma LAG3 levels were found to be the independent predictors of eGFR adjusted for age and gender (p = 0.016) Conclusions: Altogether, the studies involved in this thesis identified increased expression of urinary exosomal miR-21-5p and decreased expression of urinary exosomal miR-30b5p and plasma miR-190a-5p in individuals with T2DKD. Our data suggests that the altered expression profiles of circulating/exosomal miRNAs may prove useful in the detection of T2DKD. However, there are still potential confounding factors that influence miRNA expression and the difficulties associated with the determination of the source of the circulating miRNAs. Technology limitations include a lack of reliable methods for the extraction, quantification and validation of exosomal miRNAs. Therefore, more validation studies are required to confirm miRNA signatures for a better understanding of their role in T2DKD. Moreover earlier and longitudinal evaluation of at risk individuals of T2DKD with standardised methods may better inform the utility of these miRNAs in prediction, risk stratification and treatment of DKD. In addition, we found significant association between plasma LAG3 protein levels and renal function in patients with T2DKD, despite a lack of genetic variation in the LAG3 gene associated with T2DKD. This finding may potentially implicate the immune pathway in the development of T2DKD, but genetic variants within the coding regions of the LAG3 gene are not responsible for any of the observed variation in LAG3 protein levels associated with DKD. Further analysis of LAG3 including functional studies is necessary for clarification of our findings. NGS could facilitate screening of the whole LAG3 gene, which might shed light on the role of LAG3 and the immune pathway in T2DKD through the identification of associated variants.