Ping-Sheng Chen

MicroRNA-29c in urinary exosome/microvesicle as a biomarker of renal fibrosis

Micro (mi)RNAs are frequently dysregulated in the development of renal fibrosis. Exosomes are small membrane vesicles that could be isolated from urine secreted from all nephron segments. Here we sought to observe for the first time whether miRNA in urine exosome could serve as a potential biomarker of renal fibrosis. Urine samples were collected from 32 chronic kidney disease (CKD) patients who underwent kidney biopsy and 7 controls. Exosome was isolated and confirmed by immunogold staining of exosome marker. Members of miR-29, miR-200, and RNU6B as endogenous control were detected by RT quantitative PCR. Electronic microscopy verified a typical shape of exosome with average size of 65.1 nm and labeled it with anti-CD9 and anti-aquaporin 2 antibody. Members of miR-29 and miR-200 are readily measured with reduced levels compared with controls (P < 0.05) and can robustly distinguish CKD from controls [area under the curve (AUC) varied from 0.902 to 1 by receiver operating characteristics analysis]. miR-29c correlated with both estimated glomerular filtration rate (r = 0.362; P < 0.05) and degree of tubulointerstitial fibrosis (r = -0.359; P < 0.05) for CKD patients. Moreover, miRNA in exosome was decreased in mild fibrosis group compared with moderated to severe group. miR-29a and miR-29c could predict degree of tubulointerstitial fibrosis with AUC of 0.883 and 0.738 (P < 0.05). The sensitivity and specificity for distinguishing mild from moderate to severe fibrosis were 93.8 and 81.3% with the use of miR-29a and 68.8 and 81.3% for miR-29c. Overall, miR-29c in urinary exosome correlates with both renal function and degree of histological fibrosis, suggesting it as a novel, noninvasive marker for renal fibrosis.

CD2AP mRNA in urinary exosome as biomarker of kidney disease.

AIMS Podocyte injury plays an important role in the pathogenesis of kidney disease. Urinary exosomes are microvesicles released by tubular epithelial cells and podocytes containing information of their originated cells. This study investigated for the first time whether podocyte related mRNA in urinary exosome could serve as novel biomarkers for kidney disease. METHODS Urine samples were collected from 32 patients of kidney disease who underwent kidney biopsy and 7 controls. CD2AP, NPHS2 and synaptopodin were detected by real-time RT-PCR on RNA isolated from urinary exosome. RESULTS The pellet microvesicles were positively stained with exosome and podocyte marker, AQP2, CD9 and nephrin. CD2AP mRNA was lower (p=0.008) in kidney disease patients compared with controls and decreased with the increasing severity of proteinuria (p=0.06). CD2AP correlated with serum creatinine (r=-0.373, p=0.035), BUN (r=-0.445, p=0.009) and eGFR (r=0.351, p=0.046). Neither NPHS2 nor synaptopodin correlated with parameters of renal function. CD2AP mRNA correlated negatively with 24 hour urine protein (r=-0.403, p=0.022), severity of tubulointerstitial fibrosis (r=-0.394, p=0.026) and glomerulosclerosis (r=-0.389, p=0.031) and could discriminate kidney disease from controls with AUC of 0.821 (p=0.008). CONCLUSIONS Urinary exosome mRNA of CD2AP might be a non-invasive tool for detecting both renal function and fibrosis of kidney disease.

Reactive oxygen species released from hypoxic hepatocytes regulates MMP-2 expression in hepatic stellate cells.

Hypoxia is a common environmental stress factor and is associated with fibrogenesis. Matrix metalloproteinase-2 (MMP-2), produced by hepatic stellate cells (HSCs), plays an important role in liver fibrogenesis. However, inconsistent results have been reported on the impact of hypoxia on MMP-2 expression and activity in HSCs. We speculated that cell–cell interaction is involved in the regulation of MMP-2 expression and activity at low oxygen level in vivo. Therefore, in this report we investigated the mechanism by which hypoxic hepatocytes regulates MMP-2 expression in HSCs. Our results showed that the conditioned medium from hypoxia-treated rat hepatocytes strongly induced the expression of MMP-2 mRNA and protein in rat HSC-T6 cells. Reduced glutathione neutralized ROS released from hypoxic hepatocytes, leading to reduced MMP-2 expression in HSC-T6 cells. In addition, phospho-IκB-α protein level was increased in HSC-T6 cells treated with hypoxia conditioned medium, and NF-κB signaling inhibitor inhibited MMP-2 expression in HSC-T6 cells. Taken together, our data suggest that ROS is an important factor released by hypoxic hepatocytes to regulate MMP-2 expression in HSCs, and NF-κB signaling is crucially involved in ROS-induced MMP-2 expression in HSCs. Our findings suggest that strategies aimed at antagonizing the generation of ROS in hypoxic hepatocytes and inhibiting NF-κB signaling in HSCs may represent novel therapeutic options for liver fibrosis.

Late SV40 factor: A key mediator of Notch signaling in human hepatocarcinogenesis

AIM To investigate the relationship between late SV40 factor (LSF) and Notch signaling in the development and progress of hepatocellular carcinoma (HCC). METHODS Liver cancer tissue specimens from 25 patients were analyzed for Notch-1 and LSF expression by immunohistochemistry. The correlation between expression and the biological effects of Notch-1 and LSF were analyzed using genetic and pharmacological strategies in HCC cell lines and human normal cell lines, including hepatic stellate cells (HSC) and human embryonic kidney epithelial cells (HEK). RESULTS Immunohistochemistry showed that both Notch-1 and LSF were significantly upregulated in HCC samples (76%, 19/25, P < 0.0001 and 84%, 21/25, P < 0.0001, respectively) compared with non-cancer samples. Activation of Notch-1 by exogenous transfection of Notch1 intracellular domain increased LSF expression in HSC and HEK cells to levels similar to those seen in HepG2 cells. Furthermore, blocking Notch-1 activation with a γ-secretase inhibitor, DAPT, downregulated LSF expression in HepG2 cells. Additionally, a biological behavior assay showed that forced overexpression of LSF promoted HepG2 cell proliferation and invasion. CONCLUSION LSF is a key mediator of the Notch signaling pathway, suggesting that it might be a novel therapeutic target for the treatment of HCC.

Urinary Vimentin mRNA as a Potential Novel Biomarker of Renal Fibrosis

Renal fibrosis is a histological outcome of chronic kidney disease (CKD) progression. However, the noninvasive detection of renal fibrosis remains a challenge. Here we constructed a renal fibrosis target mRNA array and used it to detect urinary mRNAs of CKD patients for investigating potential noninvasive biomarkers of renal fibrosis. We collected urine samples from 39 biopsy-proven CKD patients and 11 healthy controls in the training set. Urinary mRNA profiles of 86 genes showed a total of 21 mRNAs that were differentially expressed between CKD patients and controls (P < 0.05), and vimentin (VIM) mRNA demonstrated the highest change fold of 9.99 in CKD vs. controls with robust correlations with decline of renal function and severity of tubulointerstitial fibrosis. Additionally, VIM mRNA further differentiated patients with moderate-to-severe fibrosis from none-to-mild fibrosis group with an area of the curve of 0.796 (P = 0.008). A verification of VIM mRNA in the urine of an additional 96 patients and 20 controls showed that VIM is not only well correlated with renal function parameters but also correlated with proteinuria and renal fibrosis scores. Multiple logistic regression and receiver-operating characteristics analysis further showed that urine VIM mRNA is the best predictive parameter of renal fibrosis compared with estimated glomerular filtration rate, serum creatinine, and blood urea nitrogen. In addition, there is no improved predictive performance for the composite biomarkers to predict renal fibrosis severity compared with a single gene of VIM. Overall, urinary VIM mRNA might serve as a novel independent noninvasive biomarker to monitor the progression of kidney fibrosis.

TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation caused by proteinuria

Proteinuria is an important risk factor for chronic kidney diseases (CKD). Several studies have suggested that proteinuria initiates tubulointerstitial inflammation, while the mechanisms have not been fully understood. In this study, we hypothesized whether the activation of the TLR2-MyD88-NF-κB pathway is involved in tubulointerstitial inflammation induced by proteinuria. We observed expression of TLR2, MyD88, NF-κB, as well as TNF-α and IL-6 detected by immunohistostaining, Western blotting and real-time PCR in albumin-overloaded (AO) nephropathy rats. In vitro, we observed these markers in HK-2 cells stimulated by albumin. We used TLR2 siRNA or the NF-κB inhibitor BAY 11-7082 to observe the influence of TNF-α and IL-6 expression caused by albumin overload. Finally, we studied these markers in non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria. It was demonstrated that expression of TLR2, MyD88 and NF-κB were significantly increased in AO rats and in non-IgA MsPGN patients with high levels of proteinuria, and TNF-α and IL-6 expressions were increased after NF-κB activation. Furthermore, TNF-α and IL-6 expression was positively correlated with the level of proteinuria. Albumin-overload induced TNF-α and IL-6 secretions by the TLR2-MyD88-NF-κB pathway activation, which could be attenuated by the TLR2 siRNA or BAY 11-7082 in HK-2 cells. In summary, we demonstrated that proteinuria may exhibit an endogenous danger-associated molecular pattern (DAMP) that induces tubulointerstitial inflammation via the TLR2-MyD88-NF-κB pathway activation.

Feature selection and classification of urinary mRNA microarray data by iterative random forest to diagnose renal fibrosis: a two-stage study

Renal fibrosis is a common pathological pathway of progressive chronic kidney disease (CKD). However, kidney function parameters are suboptimal for detecting early fibrosis, and therefore, novel biomarkers are urgently needed. We designed a 2-stage study and constructed a targeted microarray to detect urinary mRNAs of CKD patients with renal biopsy and healthy participants. We analysed the microarray data by an iterative random forest method to select candidate biomarkers and produce a more accurate classifier of renal fibrosis. Seventy-six and 49 participants were enrolled into stage I and stage II studies, respectively. By the iterative random forest method, we identified a four-mRNA signature in urinary sediment, including TGFβ1, MMP9, TIMP2, and vimentin, as important features of tubulointerstitial fibrosis (TIF). All four mRNAs significantly correlated with TIF scores and discriminated TIF with high sensitivity, which was further validated in the stage-II study. The combined classifiers showed excellent sensitivity and outperformed serum creatinine and estimated glomerular filtration rate measurements in diagnosing TIF. Another four mRNAs significantly correlated with glomerulosclerosis. These findings showed that urinary mRNAs can serve as sensitive biomarkers of renal fibrosis, and the random forest classifier containing urinary mRNAs showed favourable performance in diagnosing early renal fibrosis.

Urinary Exosomes and Exosomal CCL2 mRNA as Biomarkers of Active Histologic Injury in IgA Nephropathy

IgA nephropathy (IgAN) features variable renal pathology and a heterogeneous clinical course. Our aim was to search noninvasive biomarkers from urinary exosomes for IgAN patients; membrane nephropathy and minimal change disease were included as other glomerulopathy controls. Transmission electron microscopy and nanoparticle tracking analysis confirmed the size and morphology characteristic of urinary exosomes. Exosome markers (Alix and CD63) as well as renal cell markers [aquaporin 2 (AQP2) and nephrin] were detected, which indicate the renal origin of urinary exosomes. Exosome excretion was increased markedly in IgAN patients compared with controls and correlated with levels of proteinuria and tubular injury. More important, urinary exosome excretion correlated with greater histologic activity (mesangial hypercellularity, crescents, and endocapillary hypercellularity). Profiling of the inflammation-related mRNA revealed that exosomal chemokine (C-C motif) ligand 2 (CCL2) was up-regulated in IgAN patients. In a validation study, CCL2 was exclusively highly expressed in IgAN patients compared with healthy controls as well as minimal change disease and membrane nephropathy patients. Also, a correlation between exosomal CCL2 and estimated glomerular filtration rate levels was found in IgAN. Exosomal CCL2 was correlated with tubulointerstitial inflammation and C3 deposition. High CCL2 levels at the time of renal biopsy were associated with subsequent deterioration in renal function. Thus, urinary exosomes and exosomal CCL2 mRNA are promising biomarkers reflecting active renal histologic injury and renal function deterioration in IgAN.

Effects of Hyperbaric Oxygen with Free Radical Antagonists on the Expression of Extracellular Matrix and Matrix Metalloproteinase -2 in Rat Liver Treated by CCl4

AIM: To investigate the effects of hyperbaric oxygen (HBO) and free radical antagonists (FRA) on the expression of extracellular matrix and matrix metalloproteinase -2 in rat liver treated by CCl4, and to evaluate the preventional effectiveness on liver fibrogenesis. METHODS: Liver fibrosis model was induced by CCl4 in Wistar rats. The rats were randomly divided into control, CCl4, CCl4 with HBO treatment, CCl4 with FRA, and CCl4 with HBO and FRA groups. The rats in the latter three groups were treated with HBO or/and FRA at the 2nd and 8th weeks of the experiment. All the rats were sacrificed at the end of the 8th week. The parameters in sera and tissues were detected. RESULTS: The serum levels of AST and ALT,and the contents of hyaluronate (HA) in the group of CCl4 with HBO and FRA treatment were lower than those in the CCl4 group. The expression of collagen I, collagen IV, MMP-2 , TGF-β1 proteins and collagen I, MMP-2 mRNAs in hepatic tissues of the group was also lower than that in the CCl4 group. However ,the levels of MMP-2 activity were all higher in three groups treated with HBO or/and FRA than that in the CCl4 group. CONCLUSIONS: This work suggests that hyperbaric oxygen with free radical antagonists can delay the development of liver fibrosis.