Urinary Podocyte-Associated mRNA profile in Various Stages of Diabetic Nephropathy

Citation: Zheng M, Lv L-L, Ni J, Ni H-F, Li Q, et al. ( Urinary Podocyte-Associated mRNA profile in Various Stages of Diabetic Nephropathy Min Zheng 0 1 Lin-Li Lv 0 1 Jie Ni 0 1 Hai-Feng Ni 0 1 Qing Li 0 1 Kun-Ling Ma 0 1 Bi-Cheng Liu 0 1 Jean-Claude Dussaule, INSERM, France 0 Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine , Nanjing , China 1 We thank Mr Dai Hou-Yong (Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine) for his work in collecting samples Background: Podocyte injury and subsequent excretion in urine play a crucial role in the pathogenesis and progression of diabetic nephropathy (DN). Quantification of messenger RNA (mRNA) expression in urinary sediment by real-time PCR is emerging as a noninvasive method of screening DN-associated biomarkers. We hypothesized that the urinary mRNA profile of podocyte-associated molecules may provide important clinical insight into the different stages of diabetic nephropathy. Methods: DN patients (N = 51) and healthy controls (N = 13) were enrolled in this study. DN patients were divided into a normoalbuminuria group (UAE,30 mg/g, n = 17), a microalbuminuria group (UAE 30,300 mg/g, n = 15), and a macroalbuminuria group (UAE.300 mg/g, n = 19), according to their urinary albumin excretion (UAE). Relative mRNA abundance of synaptopodin, podocalyxin, CD2-AP, a-actin4, and podocin were quantified, and correlations between target mRNAs and clinical parameters were examined. Results: The urinary mRNA levels of all genes studied were significantly higher in the DN group compared with controls (p,0.05), and mRNA levels increased with DN progression. Urinary mRNA levels of all target genes positively correlated with both UAE and BUN. The expression of podocalyxin, CD2-AP, a-actin4, and podocin mRNA correlated with serum creatinine (r = 0.457, p = 0.001; r = 0.329, p = 0.01; r = 0.286, p = 0.021; r = 0.357, p = 0.006, respectively). Furthermore, podocalyxin mRNA was found to negatively correlate with eGFR (r = 20.349, p = 0.01). Conclusion: The urinary mRNA profiles of synaptopodin, podocalyxin, CD2-AP, a-actin4, and podocin were found to increase with the progression of DN, which suggested that quantification of podocyte-associated molecules will be useful biomarkers of DN. - Diabetic nephropathy (DN) is now the leading cause of endstage renal disease (ESRD) in patients beginning renal dialysis in the United States, and this trend is extending to developing countries as well [1,2]. The pathogenesis of DN is complex and has not yet been fully elucidated. Recent studies have shown that renal podocyte injury is pathogenically and prognostically important in DN progression. The potential mechanisms of podocyte injury include foot process effacement, hypertrophy, detachment, apoptosis, and perhaps epithelial-to-mesenchymal transition (EMT), and these mechanisms are believed to be associated with the onset and progression of DN [36]. Accumulating evidence suggests that podocyte-associated proteins or genes may correlate with proteinuria and renal function [710]. These findings bring up the interesting possibility that screening for podocyte-related molecules might be a novel strategy in monitoring the progression of DN. Currently, renal pathological examination is the gold standard for evaluating podocytopathy in DN. However, due to the invasive nature of renal biopsy, it is impractical for physicians to closely monitor patients using this method. As a technique, real-time PCR has the benefits of excellent sensitivity, quantification, and reproducibility, and it is able to measure low-abundance genes from even one single cell [11]. In recent years, with the development of reliable RNA extraction methods from urinary sediment and real-time quantitative PCR applications, the quantification of mRNA expression in urinary sediment has become an emerging modality for studying renal pathology. Some preliminary studies suggest that the determination of urinary mRNA levels might be valuable in monitoring the progression of renal disease [1215]. However, the exact clinical relevance of urinary mRNA levels remained to be determined. In this study, the expression of podocyte-associated genes in urinary sediment and their relation to disease severity were investigated in patients with DN. Clinical data The primary clinical and laboratory characteristics of the study subjects are summarized in Table 1. The age of participants in the #Macroalbuminuria, Microalbuminuria, and Normoalbuminuria vs. Healthy controls, p,0.05. *Macroalbuminuria vs. Microalbuminuria, Normoalbuminuria and Healthy controls, p,0.05. doi:10.1371/journal.pone.0020431.t001 control group was lower than in the three DN groups (p,0.05), while the differences among the DN patients were not significant. The macroalbuminuria group had a significant increase in serum creatinine and BUN compared with the other three groups (p,0.001). Urinary mRNA expr (...truncated)