A Pilot Trial Assessing Urinary Gene Expression Profiling with an mRNA Array for Diabetic Nephropathy

Min Zheng,Xiang-Dong Lei,Lin-Li Lv,Yu-Han Cao,Jie Ni,Hong Liu,Hou-Yong Dai,Dan Liu,Bi-Cheng Liu,John E Mendelson

doi:10.1371/journal.pone.0034824

Min Zheng, Xiang-Dong Lei + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0034824

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Abstract

BackgroundThe initiation and progression of diabetic nephropathy (DN) is complex. Quantification of mRNA expression in urinary sediment has emerged as a novel strategy for studying renal diseases. Considering the numerous molecules involved in DN development, a high-throughput platform with parallel detection of multiple mRNAs is needed. In this study, we constructed a self-assembling mRNA array to analyze urinary mRNAs in DN patients with aims to reveal its potential in searching novel biomarkers.MethodsmRNA array containing 88 genes were fabricated and its performance was evaluated. A pilot study with 9 subjects including 6 DN patients and 3 normal controls were studied with the array. DN patients were assigned into two groups according to their estimate glomerular rate (eGFR): DNI group (eGFR>60 ml/min/1.73 m2, n = 3) and DNII group (eGFR<60 ml/min/1.73 m2, n = 3). Urinary cell pellet was collected from each study participant. Relative abundance of these target mRNAs from urinary pellet was quantified with the array.ResultsThe array we fabricated displayed high sensitivity and specificity. Moreover, the Cts of Positive PCR Controls in our experiments were 24±0.5 which indicated high repeatability of the array. A total of 29 mRNAs were significantly increased in DN patients compared with controls (p<0.05). Among these genes, α-actinin4, CDH2, ACE, FAT1, synaptopodin, COL4α, twist, NOTCH3 mRNA expression were 15-fold higher than those in normal controls. In contrast, urinary TIMP-1 mRNA was significantly decreased in DN patients (p<0.05). It was shown that CTGF, MCP-1, PAI-1, ACE, CDH1, CDH2 mRNA varied significantly among the 3 study groups, and their mRNA levels increased with DN progression (p<0.05).ConclusionOur pilot study demonstrated that mRNA array might serve as a high-throughput and sensitive tool for detecting mRNA expression in urinary sediment. Thus, this primary study indicated that mRNA array probably could be a useful tool for searching new biomarkers for DN.

Highlights

Diabetic nephropathy (DN) is a serious complication of diabetes with high morbidity and mortality
This discordance between changes in glomerular filtration rate (GFR) and albumin excretion rate (AER) has resulted in a search for new markers that identify people with diabetes who are at risk of declining GFR independent of progressive increases in AER [3]
The Positive PCR Controls (PPC) contains artificial DNA template which has no homology to the DNA of the samples and a validated primer pair which can be used to check plate-toplate and run-to-run reproducibility of the array

Summary

Introduction

Diabetic nephropathy (DN) is a serious complication of diabetes with high morbidity and mortality It has become the most common cause of end-stage kidney disease in the world [1]. Approximately 20% of people with type 2 diabetes develop at least stage 3 CKD, defined as an estimated GFR (eGFR) less than 60 ml/min/ 1.73 m2, while remaining normoalbuminuric. This discordance between changes in GFR and AER has resulted in a search for new markers that identify people with diabetes who are at risk of declining GFR independent of progressive increases in AER [3]. We constructed a self-assembling mRNA array to analyze urinary mRNAs in DN patients with aims to reveal its potential in searching novel biomarkers

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Conclusion