Validation of candidate mRNAs and miRNAs as biomarkers of diabetic nephropathy in a North-American cohort of type 1 and type 2 diabetes patients

Abstract

Diabetic kidney disease (DKD) is a major cause of end-stage renal disease worldwide. In humans, the study of potentially pathways associated with its development and progression is complicated by the need for renal biopsy. In recent years, the availability of more sensitive techniques of molecular biology has increased the use of urine in the study of renal diseases. Besides being an organ-specific sample, urine is an easily obtainable material which does not require prior patient preparation for its collection. We conducted urine collection of type 1 diabetes (T1D) patients in order to identify new markers that could predict renal function decline and to better understand the pathogenesis of DKD and found increased mRNA expression of the gene encoding thioredoxin interacting protein (TXNIP) in the urinary sediment of patients presenting a decline in estimated glomerular filtration rate (eGFR)> 5 mL/min/1.73m2 after a 2-year median follow-up ("decliners") compared to patients "nondecliners". As a next step, we performed a RNA sequencing experiment using urinary sediment of healthy controls, decliners and nondecliners T1D patients to evaluate the differential gene expression profile of the urinary sediment among the three groups. From 436 mRNAs that were differentially expressed (p<0.05), we selected the top 10 genes that were increasingly up- or down-regulated according to renal function worsening. Nonetheless, elucidation of the translational value of the gene signatures requires validation in large cohorts of patients with detailed clinical history and demographic details. Besides mRNA, micro RNAs (miRNAs) are short non-coding RNAs that have been studied in DRD and could lead to the identification of novel biomarkers and therapeutic targets. Recently a new signature of 9 miRNAs that achieved high internal validity for the future development of microalbuminuria was published. Despite interesting, this study was conducted in a relative small number of patients (n= 27), making new studies necessary to corroborate their early observations in larger cohorts. Taking into account the data available on genes encoding mRNA and miRNAs as potential signatures for DKD, the Laboratory of kidney toxicology and Regeneration at Harvard Medical School is collecting urine samples from T1D and type 2 diabetes (T2D) patients. Three hundred and ten urine samples from T1D and T2D is available for validation of mRNA and miRNA studies. Also, the laboratory has 60 samples of human kidney tissue from biopsies of T1D patients with chronic kidney disease. Thus, the main objective of this project is to confirm the expression of the candidate miRNAs published on the literature and of mRNAs found in our RNA seq experiment. Additionally, we intend to evaluate at the protein level, the expression of some of the candidate genes in human kidney tissue from T1D patients as potential biomarkers of DRD. Urine samples will be used for miRNA and mRNA isolation with the use of commercially available kits, followed by cDNA synthesis and quantification by qPCR. Paraffin-embedded kidney tissues obtained from patients with biopsy-proven DKD will be used for protein expression analyses by immunofluorescence. The results from this study can contribute to the identification of a mRNA and a miRNA signature for patients with different stages of DKD. As a final outcome, the availability of clinical data will provide the opportunity to study the association of the potential biomarkers with progression and regression of microalbuminuria and its correlation with GFR decline. (AU)