Background: The 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.
Methods: mRNA 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 m(2), n = 3) and DNII group (eGFR<60 ml/min/1.73 m(2), 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.
Results: The 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).
Conclusion: Our 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.