Wen-Yan Huang

254C>G: a TRPC6 promoter variation associated with enhanced transcription and steroid-resistant nephrotic syndrome in Chinese children

Background:Mutations in canonical transient receptor potential channel 6 (TRPC6) have been identified as responsible for the development of focal segmental glomerulosclerosis, a proteinuric disease with steroid resistance and poor prognosis. This study explores the prevalence of TRPC6 variants in Chinese children with idiopathic nephrotic syndrome (INS), the genotype/phenotype correlation of TRPC6 variants, the therapeutic response, and the underlying molecular mechanism.Methods:Fifty-one children with sporadic INS were enrolled: 23 steroid-sensitive cases and 28 steroid-resistant cases Polymerase chain reaction was used to amplify 13 exons and the promoter sequences of TRPC6 before sequencing. The expression of TRPC6 in renal tissues was illustrated by immunohistochemistry staining. The transcriptional activity of variants in TRPC6 promoter was measured by the luciferase assay.Results:Three variants (-254C>G, rs3824934; +43C/T, rs3802829; and 240 G>A, rs17096918) were identified. The allele frequency of the -254C>G single-nucleotide polymorphism (SNP) in the steroid-resistant nephrotic syndrome (SRNS) patients (40.5%) was higher than that in the steroid-sensitive nephrotic syndrome subjects (27.1%; P = 0.046). The -254C>G SNP enhanced transcription from TRPC6 promoter in vitro and was associated with increased TRPC6 expression in renal tissues of SRNS patients.Conclusion:-254C>G, a SNP underlying enhanced TRPC6 transcription and expression, may be correlated with the development of steroid resistance in Chinese children with INS.

The expression of response gene to complement 32 on renal ischemia reperfusion injury in rat.

Abstract To investigate the expression of response gene to complement 32 (RGC32) in rat with acute kidney injury (AKI) and to explore the role of RGC32 in renal injury and repair induced by ischemia reperfusion. Rats were randomly divided into two groups, including sham operation group (n = 48) and acute ischemia reperfusion injury (IRI) group (n = 48). Rats were sacrificed following reperfusion 2 h, 6 h, 24 h, 48 h, 72 h, 1 week (w), 2 w, and 4 w. The distribution and expression of RGC32 in renal tissue were observed by means of immunohistochemistry. The mean density of the images detected by Image-Pro Plus 6 was designated as the representative RGC32 expression levels. Meanwhile, RGC32 mRNA expression was measured by qPCR. RGC32 mainly expressed in cytoplasm of proximal tubular epithelial cells. However, RGC32 did not express in renal interstitium and vessels. The expression levels of RGC32 measured by immunohistochemistry at different reperfusion time were 0.0168 ± 0.0029, 0.0156 ± 0.0021, 0.0065 ± 0.0013, 0.0075 ± 0.0013, 0.0096 ± 0.0014, 0.0132 ± 0.0016, 0.0169 ± 0.0014, 0.0179 ± 0.0022, respectively. Compared with the sham group, the level of RGC32 expression in IRI group was significant lower at 24 h, 48 h, 72 h after IRI (p < 0.05). The expression levels of RGC32 mRNA at different reperfusion time measured by qPCR were corroborated the immunohistochemistry finding. The in vitro experiments show the expression of α-SMA and extracellular matrix expression increased signification when the RGC32 was silenced. Our data showed that the RGC32 expression in AKI rat decreased significantly reduces with different reperfusion time and performs a time-dependent manner. RGC32 may play an important role in the pathogenesis of AKI following IRI and repair in rat.

Expression of response gene to complement-32 in renal tissue of children with immunoglobulin A nephropathy

Abstract Objective. This study aimed to investigate the expression and significance of response gene to complement-32 (RGC-32) in renal tissue of children with immunoglobulin A nephropathy (IgAN). Material and methods. Forty-five patients diagnosed as having IgAN by renal biopsy were enrolled. The expression of RGC-32, α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) was observed by immunohistostaining. The relationshis between the expression of RGC-32, α-SMA, TGF-β1, degree of renal pathological lesions in IgAN and clinical index were assessed by Spearman correlation. Results. Immunohistostaining analysis showed that RGC-32 protein was present in epithelial cells of renal tubules in normal and IgAN renal tissues. With more severe renal pathological lesions, the expression of RGC-32 in IgAN was increased. The expression of RGC-32 was positively correlated with that of α-SMA, TGF-β1 and the degree of renal pathological lesions in children with IgAN (p < 0.05), but had no relationship with serum creatinine, urinary N-acetyl-β-d-glucosaminidase/creatinine, urinary microalbuminuria/creatinine, urinary microimmunoglobulin/creatinine or urinary α1-microglobulin/creatinine ratio (p > 0.05). Conclusion. Expression of RGC-32 can reflect the degree of renal pathological lesions in IgAN. RGC-32 may participate in the renal tubulointerstitial lesions in children with IgAN, especially in epithelial –mesenchymal transition induced by TGF-β1.

Response gene to complement 32 regulates the G2/M phase checkpoint during renal tubular epithelial cell repair

BackgroundThe aim of this study was to evaluate the influence of RGC-32 (response gene to complement 32) on cell cycle progression in renal tubular epithelial cell injury.MethodsNRK-52E cells with overexpressed or silenced RGC-32 were constructed via transient transfection with RGC-32 expression plasmid and RGC-32 siRNA plasmid, and the cell cycle distribution was determined. The expression levels of fibrosis factors, including smooth muscle action (α-SMA), fibronectin (FN) and E-cadherin, were assessed in cells with silenced RGC-32.ResultsThe cells were injured via TNF-α treatment, and the injury was detectable by the enhanced expression of neutrophil gelatinase-associated lipocalin (NGAL). RGC-32 expression also increased significantly. The number of cells at G2/M phase increased dramatically in RGC-32 silenced cells, indicating that RGC-32 silencing induced G2/M arrest. In addition, after treatment with TNF-α, the NRK-52E cells with silenced RGC-32 showed significantly increased expression of α-SMA and FN, but decreased expression of E-cadherin.ConclusionsThe results of this study suggest that RGC-32 probably has an important impact on the repair process of renal tubular epithelial cells in vitro by regulating the G2/M phase checkpoint, cell fibrosis and cell adhesion. However, the exact mechanism needs to be further elucidated.

PS-235 The Effects Of Response Gene To Complement 32 As A New Biomarker In Children With Acute Kidney Injury

Background and aims To investigate the new biomarkers of acute kidney injury, as well as to conform the values of response gene to complement-32 (RGC-32) protein for early diagnosis of acute kidney injury in children who had undergone cardiopulmonary bypass (CPB). Methods 67 patients accepted CPB assigned to acute kidneyinjury group (AKI group) or non-acute kidney injury group (non-AKI group). Serum samples were taken regularly after CPB 30 min, 2 h, 4 h, 24 h, 48 h and 72 h for serumRGC-32, creatinine (Scr) and Cystatin C (CysC) measurement. Results The incidence of AKI was 34%, including 15 cases with Risk stage AKI, 4 cases with Injury stage AKI, 3 cases with Failure stage AKI, 1 cases with Loss stage AKI. The values for sensitivity of serum RGC-32 after CPB 30 min, 2 h and 4 h as 0.914, 0.824, 0.824 and the values for specificity of serum RGC-32 as 0.619, 0.667, 0.810, respectively. Conclusion In this study, our results first identify that possibly the sensitivity of serum RGC-32 for detecting AKI are much higher than that of Scr and serum CysC in children who had accepted CPB, and that RGC-32 may be a new biomarker for early detection of AKI. However, the conclusion needs to be further elucidated.