A pilot trial assessing urinary gene expression profiling with an mRNA array for diabetic nephropathy.
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).


CONCLUSIONS
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.
Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, China.
Min
M
Zheng
Lin-Li
LL
Lv
Yu-Han
YH
Cao
Hong
H
Liu
Jie
J
Ni
Hou-Yong
HY
Dai
Dan
D
Liu
Xiang-Dong
XD
Lei
Bi-Cheng
BC
Liu
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000652:urine; D003928:Diabetic Nephropathies / Q000175:diagnosis* / Q000235:genetics / Q000652:urine; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D008403:Mass Screening; D046228:Microarray Analysis; D008875:Middle Aged; D010865:Pilot Projects; D012333:RNA, Messenger / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015415:Biomarkers; D012333:RNA, Messenger