Lin-Li Lv

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.

Isolation and quantification of microRNAs from urinary exosomes/microvesicles for biomarker discovery.

Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×105 copies/ul for miR-200c. Exosomes was stable at 4℃ 24h for shipping before stored at -80℃ and was stable in urine when stored at -80°C for 12months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R2>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts.

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

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, α-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, α-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 = −0.349, p = 0.01). Conclusion The urinary mRNA profiles of synaptopodin, podocalyxin, CD2-AP, α-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.

Application of Antibody Array Technology in the Analysis of Urinary Cytokine Profiles in Patients with Chronic Kidney Disease

Aims: Emerging evidence suggests that the urinary excretion of cytokines is associated with the progression of chronic kidney disease (CKD). However, detection of urinary cytokines in high throughput is still a problem in clinical practice. In this cross-sectional study, we applied a novel proteomic technology, antibody array, to analyze urinary cytokine profiles in patients with CKD. Methods: A total of 10 subjects including 7 CKD patients and 3 normal controls were studied. These patients with CKD were divided into two groups according to the levels of estimated glomerular filtration rate (eGFR): group A (eGFR ≧80 ml/min/1.73 m2, n = 3) and group B (eGFR ≤40 ml/min/1.73 m2, n = 4). Urine samples taken from age and gender-matched healthy volunteers (n = 3) served as control. Differential excretion of urinary cytokines was determined by human cytokine antibody array (Raybiotech, Norcross, Ga., USA). A 2-fold change in spot intensity compared to controls was considered as significant. In order to check the reliability of the data obtained by antibody array, urinary monocyte chemoattractant protein (MCP) 1 and tumor necrosis factor (TNF) α were determined by using enzyme-linked immunosorbent assay. Results: A total of 15 cytokines varied significantly in urinary samples obtained from patients with CKD compared with normal controls. It was shown that the levels of MCP-1, RANTES, tissue inhibitor of metalloproteinase (TIMP) 1, TNF-α, vascular endothelial growth factor (VEGF), E-selectin, Fas, intercellular adhesion molecule 1, interleukin 2, matrix metalloproteinase (MMP) 2 and transforming growth factor β in patients with CKD at stage 1–2 (group A) were 2- to 5-fold higher than those in normal controls, while the excretion of MCP-1, RANTES, TIMP-1, TNF-α and VEGF was further increased in the patients with renal insufficiency (group B). However, a lower excretion of urinary vascular cell adhesion molecule 1 and platelet-derived growth factor was found in patients with CKD compared with normal controls. Impressively, the urinary MMP-9 excretion was 492-fold higher in group A and 198-fold higher in group B than that in normal controls. The levels of MCP-1 and TNF-α correlated well with the relative n-fold change seen in the antibody array experiments. The rank correlation coefficients (r values) were 0.976 (p < 0.001) and 0.939 (p < 0.001) for MCP-1and TNF-α, respectively. Conclusion: Antibody array could serve as a fast, high-throughput and sensitive tool for detecting the excretion of urinary cytokines. Our study is the first to provide an important information regarding the utility of antibody array in evaluating the role of cytokines in the initiation and progression of CKD.

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.

Activation of the Nlrp3 inflammasome by mitochondrial reactive oxygen species: A novel mechanism of albumin-induced tubulointerstitial inflammation

Albuminuria is not only an important marker of chronic kidney disease but also a crucial contributor to tubulointerstitial inflammation (TIF). In this study, we determined whether activation of the Nlrp3 inflammasome is involved in albuminuria induced-TIF and the underlying mechanisms of inflammasome activation by mitochondrial reactive oxygen species (mROS). We established an albumin-overload induced rat nephropathy model characterised by albuminuria, renal infiltration of inflammatory cells, tubular dilation and atrophy. The renal expression levels of the Nlrp3 inflammasome, IL-1β and IL-18 were significantly increased in this animal model. In vitro, albumin time- and dose-dependently increased the expression levels of the Nlrp3 inflammasome, IL-1β and IL18. Moreover, the silencing of the Nlrp3 gene or the use of the caspase-1 inhibitor Z-VAD-fmk significantly attenuated the albumin-induced increase in IL-1β and IL-18 expression in HK2 cells. In addition, mROS generation was elevated by albumin stimulation, whereas the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited Nlrp3 expression and the release of IL-1β and IL-18. In kidney biopsy specimens obtained from patients with IgA nephropathy, Nlrp3 expression was localised to the proximal tubular epithelial cells, and this result is closely correlated with the extent of proteinuria and TIF. In summary, this study demonstrates that albuminuria may serve as an endogenous danger-associated molecular pattern (DAMP) that stimulates TIF via the mROS-mediated activation of the cytoplasmic Nlrp3 inflammasome.

Improved Mitochondrial Function Underlies the Protective Effect of Pirfenidone against Tubulointerstitial Fibrosis in 5/6 Nephrectomized Rats

Dysfunctional mitochondria participate in the progression of chronic kidney disease (CKD). Pirfenidone is a newly identified anti-fibrotic drug. However, its mechanism remains unclear. Mitochondrial dysfunction is an early event that occurs prior to the onset of renal fibrosis. In this context, we investigated the protective effect of pirfenidone on mitochondria and its relevance to apoptosis and oxidative stress in renal proximal tubular cells. A remnant kidney rat model was established. Human renal proximal tubular epithelial cells (HK2) using rotenone, a mitochondrial respiratory chain complex Ι inhibitor were further investigated in vitro to examine the mitochondrial protective effect of pirfenidone. Pirfenidone protected mitochondrial structures and functions by stabilizing the mitochondrial membrane potential, maintaining ATP production and improving the mitochondrial DNA (mtDNA) copy number. Pirfenidone decreased tubular cell apoptosis by inhibiting the mitochondrial apoptotic signaling pathway. Pirfenidone also reduced oxidative stress by enhancing manganese superoxide dismutase (Mn-SOD) and inhibiting intracellular reactive oxygen species (ROS) generation, which suggested that the anti-oxidant effects occurred at least partially via the mitochondrial pathway. Pirfenidone may be effective prior to the onset of renal fibrosis because this drug exerts its anti-fibrotic effect by protection of mitochondria in renal proximal tubular cells.

Monocytes induce proximal tubular epithelial-mesenchymal transition through NF-kappa B dependent upregulation of ICAM-1

Inflammatory cell infiltration plays a key role in the pathogenesis of tubulointerstitial damage in chronic renal diseases. In addition to secreting the profibrotic cytokines, monocytes themselves have been demonstrated to be directly associated with renal fibrogenesis. However, how infiltrating monocytes interact with resident cells and the underlying mechanisms remain elusive. In this study we investigated the effects of monocytes on phenotypic changes of human proximal tubular HK‐2 cells. The typical epithelial cell morphology of HK‐2 cells disappeared after co‐culture with monocytes, accompanied by decreased E‐cadherin expression, and increased α‐SMA and fibronectin expression, suggesting that HK‐2 cells undergo epithelial–mesenchymal transition (EMT). Further analysis revealed that the effects were dependent on direct contact of the two types of cells as conditioned medium had no effects. Interestingly, administration of CD18 antibody directly inhibited this process. Furthermore, by microarray and RT‐PCR we found that NF‐kB signaling may play a role in this process and blockade of this signaling pathway in HK‐2 cells could inhibit ICAM‐1 expression and EMT phenotypes. Taken together, these findings suggest that monocytes infiltration could directly induce EMT of HK‐2 cells via upregulation ICAM‐1 through NF‐kB signaling pathway. J. Cell. Biochem. 112: 1585–1592, 2011.

Megalin/Cubulin-Lysosome-mediated Albumin Reabsorption Is Involved in the Tubular Cell Activation of NLRP3 Inflammasome and Tubulointerstitial Inflammation

Background: NLRP3 inflammasome activation is involved in albuminuria-induced renal injury. Results: The inhibition of megalin/cubilin or lysosomal cathepsin B reduced albuminuria-induced NLRP3 inflammasome activation. Conclusion: Megalin/cubilin and lysosome rupture is involved in albumin-triggered tubular injury and TI. Significance: This study provides novel insights into albuminuria-induced TI and implicates the active control of albuminuria as a critical strategy to halt the progression of CKD. Albuminuria contributes to the development and progression of chronic kidney disease by inducing tubulointerstitial inflammation (TI) and fibrosis. However, the exact mechanisms of TI in response to albuminuria are unresolved. We previously demonstrated that NLRP3 and inflammasomes mediate albumin-induced lesions in tubular cells. Here, we further investigated the role of endocytic receptors and lysosome rupture in NLRP3 inflammasome activation. A murine proteinuric nephropathy model was induced by albumin overload as described previously. The priming and activation signals for inflammasome complex formation were evoked simultaneously by albumin excess in tubular epithelial cells. The former signal was dependent on a albumin-triggered NF-κB pathway activation. This process is mediated by the endocytic receptor, megalin and cubilin. However, the silencing of megalin or cubilin inhibited the albumin-induced NLRP3 signal. Notably, subsequent lysosome rupture and the corresponding release of lysosomal hydrolases, especially cathepsin B, were observed in tubular epithelial cells exposed to albumin. Cathepsin B release and distribution are essential for NLRP3 signal activation, and inhibitors of cathepsin B suppressed the NLRP3 signal in tubular epithelial cells. Taken together, our findings suggest that megalin/cubilin and lysosome rupture are involved in albumin-triggered tubular injury and TI. This study provides novel insights into albuminuria-induced TI and implicates the active control of albuminuria as a critical strategy to halt the progression of chronic kidney disease.

Effects of angiotensin II receptor blocker on myocardial endothelial-to-mesenchymal transition in diabetic rats

BACKGROUND Emerging evidence has indicated that the endothelial-to-mesenchymal transition (EndMT) is a crucial event during early stages of cardiac fibrosis. In the present study, we first investigated the influence of Irbesartan (Irb) on myocardial EndMT in diabetic rats. METHODS Diabetic rats were divided into two groups: the diabetic group (DM) and the Irb-treated group (DM+Irb). Wistar-Kyoto rats served as controls. The pathological changes were investigated by microscopy. Immunofluorescence was performed to evaluate the co-expression of CD31 and fibroblast-specific protein 1 (FSP1). FSP1 and α-SMA expressions were detected by RT-PCR and Western blot analysis. EndMT was also studied in human aortic endothelial cells (HAECs) that had been exposed to high glucose (HG) levels. RESULTS Increased interstitial fibrosis was detected in the DM group. Double labeling revealed CD31 expression in FSP1-positive cells in the DM group, and this expression was diminished by Irb treatment (P<0.05). In vitro, we found that HG stimulated angiotensin II synthesis in HAECs. When HAECs were exposed to HG, some of the cells acquired a spindle-shaped morphology and demonstrated a loss of CD31 labeling, which was attenuated by Irb treatment. FSP1 and α-SMA mRNA and protein expression levels were markedly upregulated in diabetic rats compared to controls, and their expressions were inhibited by Irb treatment (P<0.05). CONCLUSION The results provide the novel insight that an angiotensin II receptor blocker might prevent diabetic cardiomyopathy by abrogating EndMT in diabetic rats.