CD2-associated Protein Expression Research Articles

Hypoxia-inducible factor-1α attenuates renal podocyte injury in male rats in a simulated high-altitude environment by upregulating Krüppel-like factor 4 expression.

Previous studies have shown that podocyte injury is involved in the development of proteinuria in rats under hypobaric hypoxia conditions. Prolyl hydroxylase inhibitors (PHIs) may reduce proteinuria. This study aimed to further investigate whether the protective effects of hypoxia-inducible factor 1α (HIF1α) on podocyte injury induced by hypobaric hypoxia are related to Krüppel-like factor 4 (KLF4). Rats were housed in a low-pressure oxygen chamber to simulate a high-altitude environment (5000m), and a PHI was intraperitoneally injected. Urinary protein electrophoresis was performed and the morphology of the podocytes was observed by electron microscopy. Rat podocytes were cultured under 1% O2, and siRNA was used to interfere with KLF4 expression. The protein expression levels of HIF1α, KLF4, CD2-associated protein (CD2AP) and nephrin were determined by western blotting. Compared with those in the experimental group, the rats in the intervention group on day 14 had lower urinary protein levels, increased protein expression levels of CD2AP and nephrin, and reduced podocyte injury. The results of in vitro experiments showed that the protein expression levels of KLF4, CD2AP and nephrin were greater in the PHI intervention group and lower in the HIF1α inhibitors group than in the low-oxygen group. The protein expression of CD2AP and nephrin in the siKLF4-transfected podocytes treated with PHI and HIF1α inhibitors did not differ significantly from that in the low-oxygen group. HIF1α may be involved in reducing progressive high-altitude proteinuria by regulating KLF4 expression and contributing to the repair of podocyte injury induced by hypobaric hypoxia.

CD2AP is a potential prognostic biomarker of renal clear cell carcinoma.

CD2-associated protein (CD2AP) is a podocyte-associated gene and its reduced expression is associated with the development of proteinuria and glomerulosclerosis. However, few studies have focused on the correlation between the expression and prognosis of CD2AP in renal clear cell carcinoma (ccRCC). Therefore, we aimed to assess the regulation of CD2AP expression and prognostic value in ccRCC. Multiple databases were employed to examine the expression of CD2AP in ccRCC. RT-qPCR, Western Blot and immunohistochemistry were used to validate CD2AP expression in different cell lines and tissue samples. Kaplan-Meier analysis and ROC curve analysis were performed on the predictive prognostic performance of CD2AP. COX regression was used to construct CD2AP-related prognostic models. The TIMER and TISIDB databases were used to analyze the correlation of tumor-infiltrating immune cells with gene expression, mutations, somatic copy number variation, and immune molecules. Mass spectrometry was used to detect methylation status of the promoter CpG site of CD2AP in multiple cells. We found that CD2AP expression was downregulated in ccRCC and its lower expression level was correlation with worse patient prognosis, higher tumor stage and grade and distant metastasis through analysis of databases, ccRCC cell lines and clinical tissue samples. Moreover, database and mass spectrometry techniques identified and validated cg12968598 hypermethylation as one of the key reasons for the downregulation of CD2AP expression. CD2AP expression was also associated with macrophage and neutrophil infiltration. Taken together, our results suggest that CD2AP can be used as a diagnostic and prognostic biomarker in ccRCC patients and that DNA hypermethylation plays an important role in reducing CD2AP expression.

Bta-miR-199a-3p Inhibits LPS-Induced Inflammation in Bovine Mammary Epithelial Cells via the PI3K/AKT/NF-κB Signaling Pathway.

Mastitis is characterized by inflammatory damage to mammary gland tissue, which could decline milk production and quality and significantly affect the economic benefits of ranching. MicroRNAs (miRNAs), such as miR-199a-3p, are novel therapeutic targets in inflammation, and their regulation is an effective strategy for inflammation control. Despite its importance in humans and animals, the molecular mechanism of bovine miR-199a-3p (bta-miR-199a-3p) in dairy cow mastitis and bovine mammary epithelial cell (bMEC) inflammation is unclear. In our study, a bovine mammary epithelial cell line (MAC-T) induced by lipopolysaccharide (LPS) was used as an inflammatory cell model to investigate the molecular mechanism of bta-miR-199a-3p in the MAC-T inflammatory response. bta-miR-199a-3p was up-regulated in the LPS-induced MAC-T cells, while CD2-associated protein (CD2AP) was revealed as its target gene in a double luciferase reporter gene experiment. In addition, the overexpression of bta-miR-199a-3p negatively regulated the expression of CD2AP and the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/nuclear factor kappa-B (NF-κB) signaling pathway. These subsequently inhibited the secretion of related inflammatory factors (TNF-α, IL-1β, and IL-6) and the expression of apoptotic genes (CASP3 and CASP9), thereby alleviating the LPS-challenged inflammatory response in the MAC-T cells. Silencing of bta-miR-199a-3p, however, reversed the above effects. Thus, bta-miR-199a-3p inhibits LPS-induced inflammation in bMECs by directly targeting CD2AP and regulating the PI3K/AKT/NF-κB signaling pathway. This study reveals the potential regulatory mechanism of bta-miR-199a-3p in bMEC inflammatory immune response and may serve as a useful target for the treatment of mastitis.

ANGPTL3 is involved in kidney injury in high-fat diet-fed mice by suppressing ACTN4 expression

ObjectiveWe wanted to explore how angiopoietin-like 3 (ANGPTL3) impact hyperlipidemia-induced renal injury.MethodsANGPTL3 knockout mice and wild-type C57 mice were set up in four groups (N = 5) depending on a normal or 60% high-fat diet: wild-type with normal diet (WT), angptl3-/- with normal diet (KO), wild-type + high-fat diet (WT + HF) and angptl3-/- + high-fat diet (KO + HF). The detection time points were the 9th, 13th, 17th and 21st weeks after modeling. Serum lipid and urinary protein levels of mice in each group were detected, and pathological changes in the kidney were analyzed. Moreover, the expression of ANGPTL3, α-actinin-4 (ACTN4), CD2-associated protein (CD2AP) and podocin was tested in the glomerulus by immunohistochemistry (IHC).ResultsIn the WT + HF group, hyperlipidemia and proteinuria could be observed at the 9th week and were gradually aggravated with time. Compared with WT + HF mice, the levels of serum lipids and proteinuria in KO + HF mice were significantly reduced, and the width of podocyte foot processes (FPs) fusion was also markedly improved. The IHC results suggested that in WT + HF mice, the expression of ANGPTL3 was significantly enhanced. After modeling, ACTN4 expression was markedly weakened in the glomeruli of WT + HF mice. Different to WT mice, ACTN4 expression was not observed obviously change in KO + HF mice. Compared with the normal diet group, the expression of podocin showed a decline in WT mice treated with high-fat diet and showed a significant difference from the 17th week. In addition, podocin expression in KO + HF glomeruli was also found to be weak but not significantly different from that in WT + HF glomeruli at the four time points. The expression of CD2AP showed similar results among the four groups.ConclusionANGPTL3 could play a role in the mechanism of hyperlipidemia-associated podocyte injury via ACTN4.

Circular RNA VMA21 ameliorates lung injury in septic rat via targeting microRNA-497-5p/CD2-associated protein axis

ABSTRACT Sepsis was characterized via an acute inflammatory response to infection, often accompanying by multiple organ failure, particularly lung damage. Circular RNA (circRNA) played an important role in the pathology of a variety of diseases. However, the role of circRNA in sepsis-induced lung injury (LI) remained unknown. This study was to explore the expression and role of circVMA21 in sepsis LI and the possible molecular mechanism. The results manifested circVMA21 and CD2-associated protein (CD2AP) were down-regulated in lung tissue and lipopolysaccharide (LPS)-treated BEAS-2B, while microRNA (miR)-497-5p was up-regulated. A large number of deaths in rats after surgery of 72 h were caused via cecal ligation-perforation surgery, W/D value and Bax positive cells were increased, LI was caused, cell apoptosis, tumor necrosis factor-α, Interleukin (IL)-1β and IL-6 expression were promoted and Bcl-2 positive cells were decreased. Overexpression of circVMA21 ameliorated these phenomena. In addition, LPS-induced apoptosis and inflammation of BEAS-2B cells was improved via overexpression of circVMA21, while overexpression of miR-497-5P was opposite. Apoptosis, inflammation, and oxidative damage of BEAS-2B cells were aggravated via knockdown of circVMA21, but it was reversed by knockdown of miR-497-5p or overexpression of CD2AP. Mechanistically, CircVMA21 mediated CD2AP expression through competitive adsorption of miR-497-5p. In conclusion, this work showed circVMA21 improved LI in sepsis rats by targeting miR-497-5p/CD2AP axis, suggesting that circVMA21 may be a novel therapeutic target for sepsis-induced LI.

Long non-coding RNA TTC28-AS1 attenuates high glucose-induced damage in HK-2 cells depending on the regulation of miR-320a/CD2AP axis.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD) worldwide. Emerging evidence suggests that long non-coding RNAs (lncRNAs) play crucial roles in DN pathogenesis. The purpose of the present study was to explore the role and mechanism of lncRNA tetratricopeptide repeat domain 2B antisense RNA 1 (TTC28-AS1) in DN. Cell viability and apoptosis were assessed by the Cell Counting-8 Kit (CCK-8) assay and flow cytometry, respectively. The levels of TTC28-AS1, miR-320a and CD2-associated protein (CD2AP) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-8 were gauged by enzyme-linked immunosorbent assay (ELISA). Targeted relationship between miR-320a and TTC28-AS1 or CD2AP was evaluated by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Our data indicated that high glucose (HG) induced HK-2 cell damage by the repression of cell viability and autophagy and the enhancement of cell apoptosis, fibrosis and pro-inflammatory cytokines production. TTC28-AS1 was down-regulated and miR-320a was up-regulated in HG-induced HK-2 cells. TTC28-AS1 overexpression or miR-320a knockdown alleviated HG-induced damage in HK-2 cells. MiR-320 was a molecular mediator of TTC28-AS1 in regulating HG-induced HK-2 cell damage. Moreover, TTC28-AS1 functioned as a post-transcriptional regulator of CD2AP expression by miR-320a. MiR-320a knockdown relieved HG-induced damage in HK-2 cells by up-regulating CD2AP. Our findings suggest that TTC28-AS1 attenuates HG-induced damage in HK-2 cells at least partially by targeting the miR-320a/CD2AP axis, highlighting its role as a promising therapeutic approach for DN treatment.

Transforming growth factor-β1-induced podocyte injury is associated with increased microRNA-155 expression, enhanced inflammatory responses and MAPK pathway activation.

MicroRNA-155 (miR-155) is associated with various diseases. However, the potential role of miR-155 in early glomerular disease (EGD) remains elusive. In the present study, the clinical significance of urinary miR-155 expression was explored in patients with EGD using receiver operating characteristic curve analysis. Conditionally immortalized mouse podocytes were cultured in vitro and treated with transforming growth factor-β1 (TGF-β1) at different concentrations and durations. The gene expression levels of mRNAs and miR-155 were detected using reverse transcription-quantitative PCR. Synaptopodin, CD2-associated protein (CD2AP), p38, and extracellular signal-regulated kinase (Erk) 1/2 expressions were detected using western blotting. Cell supernatants were collected for assaying tumor necrosis factor (TNF)-α and interleukin (IL)-6 concentrations using enzyme-linked immunosorbent assay. The Pearson correlation analysis was used to analyze the correlation between miR-155 levels and TNF-α or IL-6. It was found that miR-155 levels in urine have high sensitivity and specificity in the diagnosis of EGD. Time- and dose-dependent TGF-β1 treatments downregulated synaptopodin and CD2AP expression levels, and activated the p38 and Erk 1/2 pathway. However, these effects were attenuated by p38 and Erk 1/2 phosphorylation inhibitors. Additionally, TNF-α and IL-6 secretions were elevated, and their concentrations were positively correlated with the expression of miR-155 during podocyte injury. Thus, the present study indicated that miR-155 is a potential biomarker for the diagnosis of EGD, and its expression is associated with the release of pro-inflammatory cytokines and activation of mitogen-activated protein kinase (MAPK) pathway in TGF-β1-induced podocyte injury. The present study suggests that the TGF-β1/miR-155/MAPK axis is a novel target in the mechanism of EGD.

CD2AP inhibits metastasis in gastric cancer by promoting cellular adhesion and cytoskeleton assembly.

Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that the alter of the cellular cytoskeleton and intercellular adhesion. CD2‐associated protein (CD2AP) is one of the critical proteins regulating cytoskeleton assembly and intercellular adhesion. However, no study has investigated the expression and biological significance of CD2AP in gastric cancer (GC) to date. Therefore, the aim of our study was to explore if the expression of CD2AP is associated with any clinical features of GC and to elucidate the underlying mechanism. Immunohistochemistry of 620 patient tissue samples indicated that the expression of CD2AP is downregulated in DGC. Moreover, a low CD2AP level was indicative of poor patient prognosis. In vitro, forced expression of CD2AP caused a significant decrease in the migration and invasion of GC cells, whereas depletion of CD2AP had the opposite effect. Immunofluorescence analysis indicated that CD2AP promoted cellular adhesion and influenced cell cytoskeleton assembly via interaction with the F‐actin capping protein CAPZA1. Overall, the upregulation of CD2AP could attenuate GC metastasis, suggesting CD2AP as a novel biomarker for the prognosis and treatment of patients with GC.

Human podocyte injury in the early course of hypertensive renal injury.

BACKGROUNDHypertension is prevalent in the general population and is regarded as the second leading cause of renal damage and dysfunction, outnumbered only by diabetes. However, the mechanisms remain unclear.AIMTo investigate podocyte injury induced by hypertension in the early course without massive proteinuria or renal dysfunction.METHODSThe hypertension group comprised 18 patients with hypertension accompanied by microalbuminuria, diagnosed with hypertensive renal injury according to biopsy results. For a comparison of pathological changes in renal tissue, control group 1 comprised 10 healthy volunteers, and control group 2 comprised 16 patients who underwent surgery for renal trauma.RESULTSThe hypertension group had significantly higher blood pressure (P = 0.000) and microalbuminuria (P = 0.000) compared with control group 1. In the hypertension group, urinary podocytes were detected following positive staining of podocyte-specific nephrin and/or CD2-associated protein (CD2AP) in urine sediment. Podocyte foot process fusion and a significant decrease in nephrin and/or CD2AP expression in glomeruli were observed in the hypertension group compared with control group 2. This indicated that hypertension caused podocyte injury and detachment from the glomerular basement membrane, which was consistent with urinary detection of podocytes.CONCLUSIONOur results suggest that podocyturia appears early in the course of hypertensive renal injury, and may be a sensitive marker for early prediction of hypertensive renal injury.

In Vivo Expression of NUP93 and Its Alteration by NUP93 Mutations Causing Focal Segmental Glomerulosclerosis

IntroductionMutations in genes encoding nucleoporins (NUPs; components of nuclear pore complexes [NPCs]), such as NUP93, have been reported to cause steroid-resistant nephrotic syndrome (SRNS) or focal segmental glomerulosclerosis (FSGS), which often progresses to end-stage renal disease (ESRD) in childhood. The expression of NUP93 in renal or extrarenal tissues, and the mechanism by which NUP93 mutations cause this renal phenotype, remain unclear.MethodsThe expression of NUP93 in normal control kidney and in a patient with FSGS carrying NUP93 mutations was examined by immunofluorescence analysis. The expression of NUP93 in blood cells was analyzed by Western blot analysis.ResultsImmunofluorescence analysis detected NUP93 expression in nuclei of all glomerular and tubulointerstitial cells in human kidneys. Whole-exome sequencing identified a compound heterozygous NUP93 mutation comprising a novel missense mutation p.Arg525Trp, and a previously reported mutation, p.Tyr629Cys, in a patient with FSGS that developed ESRD at the age of 6 years. In the patient’s kidney, the intensity of NUP93 immunofluorescence was significantly decreased in the nuclei of both glomerular and extraglomerular cells. The expression of CD2-associated protein (CD2AP) and nephrin in the patient’s podocytes was relatively intact. The amount of NUP93 protein was not significantly altered in the peripheral blood mononuclear cells of the patient.ConclusionNUP93 is expressed in the nuclei of all the cell types of the human kidney. Altered NUP93 expression in glomerular cells as well as extraglomerular cells by NUP93 mutations may underlie the pathogenic mechanism of SRNS or FSGS.

MiR-939-5p decreases the enrichment of RNA polymerase II in the promoter region of CD2AP involved in nephrotic syndrome.

The expression changes of CD2-associated protein (CD2AP) can lead to kidney diseases with proteinuria, including nephrotic syndrome (NS). A recent study reported that miRNAs may be important transcriptional regulators. In this study, we found increased expression of miR-939-5p and decreased expression of CD2AP in the peripheral blood of patients with NS. However, miR-939-5p did not show a regulatory effect on the 3'-untranslated region of CD2AP. The expression levels of specific protein 1 and adenovirus E2 promoter-binding factor 1, important transcription regulators in the promoter region of CD2AP, were also not affected by microRNA (miR)-939-5p. We confirmed that miR-939-5p is in the nucleus by fluorescent in situ hybridization and cytoplasmic separation polymerase chain reaction. The promoter plasmid and miR-939-5p were cotransfected into HEK-293 cells, and the luciferase reporter gene assay was used to analyze the promoter activity. We found that miR-939-5p binds to a specific sequence in the CD2AP promoter. miR-939-5p was confirmed to reduce the recruitment of RNA polymerase II to the CD2AP promoter region by chromatin immunoprecipitation. These findings improve our understanding of the mechanism of miR-939-5p in NS and provide potential molecular therapeutic targets for NS.

CD2-Associated Protein Contributes to Hepatitis C, Virus Propagation and Steatosis by Disrupting Insulin Signaling.

Chronic hepatitis C virus (HCV) infection can result in steatosis, a condition displaying aberrant accumulation of neutral lipid vesicles, the component of lipid droplets (LDs), which are essential for HCV assembly. However, the interplay between HCV infection and steatosis remains unclear. Here, we show that HCV‐infected cells have higher levels of CD2‐associated protein (CD2AP), which plays two distinct, yet tightly linked, roles in HCV pathogenesis: Elevated CD2AP binds to nonstructural protein 5A (NS5A) and participates in the transport of NS5A to LDs to facilitate viral assembly; Up‐regulated CD2AP also interacts with casitas B‐lineage lymphoma (b) (Cbl/Cbl‐b) E3 ligases to degrade insulin receptor substrate 1 (IRS1), which, in turn, disrupts insulin signaling and increases LD accumulation through the IRS1/protein kinase B (Akt)/adenosine monophosphate‐activated protein kinase (AMPK)/hormone‐sensitive lipase (HSL) signaling axis to accommodate viral assembly. In the HCV‐infected mouse model, CD2AP expression is up‐regulated during the chronic infection stage and this up‐regulation correlates well with liver steatosis. Importantly, CD2AP up‐regulation was also detected in HCV‐infected human liver biopsies showing steatosis compared to non‐HCV‐infected controls. Conclusion: CD2AP is indicated as a protein up‐regulated by HCV infection, which, in turn, stimulates HCV propagation and steatosis by disrupting insulin signaling; targeting CD2AP may offer an opportunity for alleviating HCV infection and its associated liver pathology. (Hepatology 2018;XX:XXX‐XXX.)

Inhibition of SHIP2 in CD2AP-deficient podocytes ameliorates reactive oxygen species generation but aggravates apoptosis

Lack of CD2-associated protein (CD2AP) in mice increases podocyte apoptosis and leads to glomerulosclerosis and renal failure. We showed previously that SHIP2, a negative regulator of the PI3K/AKT signalling pathway, interacts with CD2AP. Here, we found that the expression level and activity of SHIP2 and production of reactive oxygen species (ROS) are increased in cultured CD2AP knockout (CD2AP−/−) mouse podocytes. Oxidative stress was also increased in CD2AP−/− mouse glomeruli in vivo. We found that puromycin aminonucleoside (PA), known to increase ROS production and apoptosis, increases SHIP2 activity and reduces CD2AP expression in cultured human podocytes. PDK1 and CDK2, central regulators of AKT, were downregulated in CD2AP−/− or PA-treated podocytes. Downregulation of PDK1 and CDK2, ROS generation and apoptosis were prevented by CD2AP overexpression in both models. Notably, inhibition of SHIP2 activity with a small molecule inhibitor AS1949490 ameliorated ROS production in CD2AP−/− podocytes, but, surprisingly, further reduced PDK1 expression and aggravated apoptosis. AKT- and ERK-mediated signalling was diminished and remained reduced after AS1949490 treatment in the absence of CD2AP. The data suggest that inhibition of the catalytic activity of SHIP2 is beneficial in reducing oxidative stress, but leads to deleterious increase in apoptosis in podocytes with reduced expression of CD2AP.

CD2-associated protein/phosphoinositide 3-kinase signaling has a preventive role in angiotensin II-induced podocyte apoptosis

Angiotensin II (Ang II) works as a paracrine or autocrine cytokine agent to regulate renal functions and promotes podocytes dysfunction directly or indirectly, causing proteinuria. The glomerular slit diaphragm (SD) serves as a size-selective barrier and is linked to the actin-based cytoskeleton by adaptor proteins, including CD2-associated protein (CD2AP). Therefore, damages to CD2AP affect not only the function of the SD, but also directly disrupt the podocyte cytoskeleton, leading to proteinuria. In addition, CD2AP can facilitate the nephrin-induced phosphoinositide 3-kinase (PI3-K)/Akt signaling, which protects podocytes from apoptosis. Here we found that CD2AP staining was located diffusely but predominantly in the peripheral cytoplasm and CD2AP co-localized with nephrin in mouse podocytes; however, Ang II decreased CD2AP staining diffusely and induced a separation from concentrated nephrin. Ang II notably reduced CD2AP expression in time- and concentration-dependent manners, and this was significantly recovered by losartan. Ang II induced podocyte apoptosis in time- and concentration-dependent manners in TUNEL and FACS assays. LY294002, a PI3-K inhibitor, further reduced CD2AP expression and increased podocyte apoptosis, which was augmented by siRNA for CD2AP. Thus, Ang II induces the relocalization and reduction of CD2AP via AT1R, which would cause podocyte apoptosis by the suppression of CD2AP/PI3-K signaling.

THE ROLE OF CD2AP IN APP PROCESSING

Genome-wide association studies (GWAS) have identified >20 risk loci for late-onset Alzheimer's disease (LOAD). Several of these risk genes are involved in endocytosis, a process believed to be disrupted during the early stages of LOAD. The CD2-associated protein (CD2AP) gene is strongly associated with LOAD risk (p=5.2x10-11) and plays a role in receptor-mediated endocytosis. We hypothesised that CD2AP contributes to amyloid precursor protein (APP) metabolism and subsequent amyloid –beta (Aβ) generation through its regulation of clathrin-mediated endocytosis. To test this, we initially characterised the effects of CD2AP knockdown on APP metabolism and Aβ production in a human neuronal cell line. A recent study found that another GWAS-identified risk gene PICALM, regulates trafficking of Aβ across the endothelial cells which form the blood brain barrier (BBB). We hypothesise that CD2AP acts in a similar way to PICALM and that knocking down CD2AP diminishes Aβ trafficking across the BBB, decreasing the amount of Aβ cleared from the brain. To investigate this, we are characterising the effects of CD2AP knockdown in an in vitro model of the BBB. Endogenous CD2AP expression was knocked down in H4 and hMEC/D3 cells using siRNA. Knockdown was quantified by western blot and Image J analysis. Levels of APP, Aβ, and APP metabolites were quantified using ELISAs. The rate of receptor-mediated endocytosis was measured by a transferrin uptake assay. Co-localisation of CD2AP with APP and endosomal markers was assessed using immunocytochemistry. Transcytosis in hMEC/D3 cells will be assessed using Transwell assays. We observed a significant 30.27±0.07 % increase in Aβ40 expression (p=0.0046). Expression levels of APP and APP processing metabolites was not significantly altered. Results from our experiments in progress will be made available at AAIC 2016. Knockdown of CD2AP expression resulted in a significant increase in extracellular amyloid-B (Aβ) levels, which did not appear to be directly related to changes in APP processing. This study is ongoing and novel data will be presented at AAIC 2016. Overall, our study provides valuable insights into how CD2AP contributes to LOAD susceptibility. Effect of CD2AP siRNA treatment of H4 cells on extracellular Aβ40 protein levels. Results are expressed as a percentage of oligofectamine only treated cells. Four biological replicates were analysed for each assay. Statistical significance of CD2AP siRNA vs. control siRNA treatment was determined by two-tailed student's t-test., **p = 0.0046; *= 0.0223 Error bars = SEM.

Cyclosporine A induced histological changes of Cathepsin L and CD2AP expression in renal glomeruli and tubules.

Cyclosporine A (CsA) is used globally as an immunosuppressant for the treatment of immune-mediated nephrotic syndrome (NS). However, its long-term use causes nephrotoxicity characterized by tubulointerstitial injury and glomerulosclerosis. The present study aimed to investigate the associations between histomorphological findings and immunohistological expression of Cathepsin L (CatL) and CD2-associated protein (CD2AP) in patients with NS mediated with CsA. A total of 18 patients with child-onset NS were divided into two groups after treatment with CsA for 2years (group A; n=10) and more than 4years (group B; n=8), respectively. Analyses of relationships between tubulointerstitial disorders and expression of CatL and CD2AP proteins were performed using immunohistochemistry of paired renal specimens. Glomeruli with arteriole hyalinization were significantly increased in both groups depending on dosage periods, although degrees of tubule and interstitial injury did not differ between groups. CD2AP expression was significantly greater in podocytes (P=0.046) and was significantly less in proximal tubule cells (P=0.014) in patients of group B compared with those of group A. Moreover, CD2AP expression was significantly increased in lateral tubule cells in both groups (group A, P=0.02; group B, P=0.001), and CatL expression in glomeruli and tubule cells did not change with the duration of CsA treatment in either patient group. CD2AP expression in renal tubules may histologically associate with tissue hypoxia and reflected recovery from CsA-mediated renal injury in patients, even with mild histological features of tubulointerstitial disorder.

MicroRNA-939 down-regulates CD2-associated protein by targeting promoter in HEK-293T cells

CD2-associated protein (CD2AP) serves as a slit diaphragm (SD) protein and plays essential roles in maintaining podocyte integrity and reducing proteinuria. MicroRNAs (miRNAs) are novel regulators of gene expression. Podocyte-specific loss of miRNAs would lead to significant proteinuria. Here, we report new evidence in which miRNAs may function to suppress CD2AP expression through a transcriptional way. By scanning human CD2AP promoter in silico for sequences complementary to known miRNAs, we chose miR-939, miR-148b*, miR-191*, miR-638 as four candidates and transfected them into HEK-293T cells. Dual-luciferase reporter assay identified that only miR-939 significantly reduced the relative luciferase activity of CD2AP promoter region. Further analysis confirmed that the mRNA and protein expressions of CD2AP were also down-regulated by miR-939. In conclusion, we have identified that miR-939 targets CD2AP promoter sequences and suppresses its gene expression. These findings suggest that miRNAs may mediate podocyte injury via reducing the expression of the SD proteins, such as CD2AP.

MicroRNA-939 regulating CD2-associated protein expression by targeting promoter

Objective To verify the targeting regulatory relationship between microRNA-939 (miR-939) and CD2-associated protein (CD2AP). Methods The online RegRNA software was used to predict the human CD2AP promoter for potential binding sites complementary to miR-939.HEK-293T cells were cotransfected with human CD2AP promoter plasmid pGL3-2K and microRNA negative control (miR-NC) or miR-939 mimics, and the relative luciferase activity(RLA) was detected at 24 h post-transfection.HEK-293T cells were transfected with miR-NC or miR-939 mimics for 48 h, and the CD2AP mRNA expression level was detected by adopting reverse transcript and real-time fluorescence quantification-PCR, while the CD2AP protein expression level was detected by using Western blot. Results (1) There were 2 miR-939 binding sites at CD2AP promoter region, located at -468 to -491 and -654 to -677 upstream of initiation codon ATG (marked as + 1) relatively.(2)At 30 nmol/L, 50 nmol/L, the RLA in miR-NC group and miR-939 group were 6.81±0.88 vs 6.07±2.24, 5.88±1.44 vs 3.94±0.79 relatively, and there were no significant differences between the 2 groups (t=3.04, 2.06, all P>0.05), while the RLA between the 2 groups were 5.58±0.58 vs 3.29±0.64 at 100 nmol/L, and the difference was significant between the 2 groups(t=4.07, P<0.05). (3) At 30 nmol/L, 50 nmol/L and 100 nmol/L, the relative CD2AP mRNA expression in miR-NC group and miR-939 group were 1.00±0.01 vs 0.80±0.08, 1.00±0.00 vs 0.80±0.13 and 1.00±0.00 vs 0.72±0.07 relatively, while the CD2AP mRNA expression was decreased by 20%-30% at each concentration level, and there were significant differences between the 2 groups (t=4.44, 2.93, 6.84, all P<0.05). (4) At 50 nmol/L, the relative CD2AP protein expression in miR-NC group and miR-939 group were 0.48±0.09 vs 0.19±0.12, and the CD2AP protein expression was decreased, and the difference was significant (t=3.36, P<0.05). Conclusions CD2AP is the target gene of miR-939, and miR-939 can down-regulate the expression of CD2AP both in mRNA and protein levels by targeting its promoter region, which indicates that miR-939 may mediate the podocyte injury. Key words: MicroRNA-939; CD2-associated protein; Podocyte; Promoter; Gene expression regulation

NF-κB-dependent increase in tissue factor expression is responsible for hypoxic podocyte injury.

Fibrin deposition within glomeruli is commonly seen in kidney biopsy specimens, suggesting enhanced coagulant activity. Tissue factor (TF) is a coagulation factor which is also related to various biological effects, and TF is upregulated by hypoxia in cancer cells. Recently, hypoxic podocyte injury has been proposed, therefore, we investigated TF expression in hypoxia. Conditionally immortalized human podocytes were differentiated and treated under hypoxic or normoxic conditions. mRNA expressions of TF and tissue factor pathway inhibitor (TFPI) were analyzed by quantitative RT-PCR. Protein levels of TF and TFPI were tested by enzyme-linked immunosorbent assay. We employed small interfering RNA (siRNA) to temporary knockdown early growth response protein 1 (Egr-1), hypoxia-inducible factor-1α (HIF-1α) and TF. The expression of CD2-associated protein (CD2AP) mRNA and phalloidin staining was examined to assess podocyte injury. Hypoxia increased mRNA expression of TF (6h: 2.3±0.05 fold, p<0.001, 24h: 5.6±2.4 fold, p<0.05) and suppressed TFPI (6h: 0.54±0.04 fold, p<0.05, 24h: 0.24±0.06 fold, p<0.001) compared with normoxia. Similarly, protein levels of TF were increased and TFPI were decreased. Egr-1 siRNA did not change TF mRNA expression. Pyrrolidine dithiocarbamate (PDTC), a nuclear factor kappa B (NF-κB) inhibitor, significantly reduced hypoxia induced TF expression, and HIF-1α knockdown further increased TF. Hypoxia resulted in decreased CD2AP and actin reorganization in podocytes, and these changes were attenuated by TF siRNA. Hypoxia increased the expression of TF in human podocytes NF-κB dependently. TF may have a critical role in the hypoxic podocyte injury.

Diabetic conditions downregulate the expression of CD2AP in podocytes via PI3-K/Akt signalling.

Proteinuria is typically accompanied by structural and compositional changes of the foot processes and of the slit diaphragms between podocytes. CD2-associated protein (CD2AP) in podocytes serves as an adaptor protein binding to nephrin and podocin, anchoring these slit diaphragm proteins to actin filaments of podocyte cytoskeleton and sending signals inward or outward. In the present study, we prepared streptozotocin-induced diabetic renal tissues and cultured podocytes in diabetic conditions to investigate podocyte phenotypical changes, including quantitative and distributional changes of CD2AP protein and search for the signalling mechanisms in diabetic conditions. We prepared cultured rat glomerular epithelial cells and mouse podocytes to study how high glucose and advanced glycosylation end products (AGE) induce phenotypical changes of cultured podocyte, under (1) normal glucose (5 mM, = control), (2) high glucose (30 mM), (3) AGE-added or (4) high glucose plus AGE-added conditions. According to diabetic duration, density of CD2AP in renal tissue of experimental diabetic nephropathy became conglomerulated and diminished. In cultured podocytes, CD2AP co-localized with nephrin and zonula occludens-1 by confocal imaging. High glucose and high glucose plus AGE induced the relocalization and concentration of CD2AP at internal cytoplasmic and perinuclear areas of podocytes. High glucose plus AGE-added condition also decreased CD2AP protein amount and its mRNA expression compared with normal glucose or osmotic control conditions. In addition, LY294002, a phosphoinositide 3-kinase inhibitor, prevented the quantitative and distributional changes of CD2AP induced by high glucose and AGE. These findings suggest that diabetic conditions induce the phenotypical changes of podocyte CD2AP possibly via phosphoinositide 3-kinase/Akt signalling.