[Primary culture and identification of mouse renal proximal tubular epithelial cells].

The present study was aimed to establish a modified method for culturing mouse renal proximal tubular epithelial cells (TECs). Renal cortex was isolated from mouse kidney and scissored into pieces. TECs were separated by digesting scissored renal cortex in type II collagenase combined with strainer filtration, and then cultured in DMEM. The morphology of TECs was observed under inverted microscopy. The cell proliferative ability was assessed by flow cytometry, and cell viability was analyzed by CCK-8 assay. The purity of TECs was identified by immunofluorescence. Immunofluorescence observation showed that more than 95% cells were epithelial marker CK18 positive and more than 90% cells expressed renal proximal TECs marker proteins, Villin, AQP1, and SGLT2. The cells could be subcultured for about 5 times. The cell proliferative ability declined following the repeated passage. This study introduced a modified efficient method for culturing highly purified mouse renal proximal TECs.

Urinary DcR2 is a novel biomarker for tubulointerstitial injury in patients with diabetic nephropathy.

Tubulointerstitial injury (TII) plays a crucial role in the progression of diabetic nephropathy (DN), but lack of specific and sensitive biomarkers for monitoring TII in DN management. This study is to investigate whether urinary decoy receptor 2 (uDcR2) could serve as a novel noninvasive biomarker for assessing TII in DN. We recruited 311 type 2 diabetics and 139 DN patients who were diagnosed by renal biopsy. uDcR2 levels were measured by ELISA, and renal DcR2 expression was detected immunohistochemically. Associations between uDcR2 and renal DcR2 and renal functional parameters were evaluated. Receiver operating characteristics (ROC) curve analyzed area under the curve (AUC) of uDcR2 for assessing TII. Double staining was undertaken for renal DcR2 with proximal and distal tubular markers; senescent markers p16, p21, and senescence-associated ß-galactosidase (SA-ß-gal); and fibrotic markers collagen I and IV. We found DcR2 was primarily expressed in renal proximal tubules; uDcR2 levels were elevated per albuminuria stratum and correlated with renal functional parameters in diabetics and were associated with percentage of tubular DcR2 and TII score in DN. The uDcR2 had an AUC of 0.909 for assessing TII in DN by ROC analysis. Almost all tubular DcR2 was coexpressed with p16 and p21, and nearly more than one-half of tubular DcR2 was positive for SA-ß-gal, primarily in collagen I- and IV-positive regions of DN. Our results indicate uDcR2 could potentially serve as a novel biomarker for TII and may reflect senescence of renal proximal tubular cells in DN pathogenesis.

Proteomics reveals defective peroxisomal fatty acid oxidation during the progression of acute kidney injury and repair.

Acute kidney injury (AKI) is characterized by a rapid decrease in renal function with high mortality and risk of progression to chronic kidney disease (CKD). Ischemia and reperfusion injury (IRI) is one of the major causes of AKI. However, the cellular and molecular responses of the kidney to IRI are complex and not fully understood. Herein, we conducted unbiased proteomics and bioinformatics analyses in an IRI mouse model on days 3, 7, and 21, and validated the results using IRI, unilateral ureteral obstruction (UUO), and biopsies from patients with AKI or CKD. The results indicated an obvious temporal expression profile of differentially expressed proteins and highlighted impaired lipid metabolism during the progression of AKI to CKD. Acyl-coenzyme A oxidase 1 (Acox1), the first rate-limiting enzyme of peroxisomal fatty acid beta-oxidation, was then selected, and its disturbed expression in the two murine models validated the proteomic findings. Accordingly, Acox1 expression was significantly downregulated in renal biopsies from patients with AKI or CKD, and its expression was negatively correlated with kidney injury score. Furthermore, in contrast to the decreased Acox1 expression, lipid droplet accumulation was remarkably increased in these renal tissues, suggesting dysregulation of fatty acid oxidation. In conclusion, our results suggest that defective peroxisomal fatty acid oxidation might be a common pathological feature in the transition from AKI to CKD, and that Acox1 is a promising intervention target for kidney injury and repair.

Decoy receptor 2 mediates the apoptosis-resistant phenotype of senescent renal tubular cells and accelerates renal fibrosis in diabetic nephropathy.

Apoptotic resistance leads to persistent accumulation of senescent cells and sustained expression of a senescence-associated secretory phenotype, playing an essential role in the progression of tissue fibrosis. However, whether senescent renal tubular epithelial cells (RTECs) exhibit an apoptosis-resistant phenotype, and the role of this phenotype in diabetic nephropathy (DN) remain unclear. Our previous study was the first to demonstrate that decoy receptor 2 (DcR2) is associated with apoptotic resistance in senescent RTECs and renal fibrosis. In this study, we aimed to further explore the mechanism of DcR2 in apoptosis-resistant RTECs and renal fibrosis in DN. DcR2 was co-localized with fibrotic markers (α-SMA, collagen IV, fibronectin), senescent marker p16, and antiapoptotic proteins FLIP and Bcl2 but rarely co-localized with caspase 3 or TUNEL. DcR2 overexpression promoted renal fibrosis in mice with streptozotocin (STZ)-induced DN, as evidenced by augmented Masson staining and upregulated expression of fibrotic markers. DcR2 overexpression also enhanced FLIP expression while reducing the expression of pro-apoptotic proteins (caspases 8 and 3) in senescent RTECs, resulting in apoptotic resistance. In contrast, DcR2 knockdown produced the opposite effects in vitro and in vivo. Moreover, quantitative proteomics and co-immunoprecipitation experiments demonstrated that DcR2 interacted with glucose-related protein 78 kDa (GRP78), which has been shown to promote apoptotic resistance in cancer. GRP78 exhibited co-localization with senescent and antiapoptotic markers but was rarely co-expressed with caspase 3 or TUNEL. Additionally, GRP78 knockdown decreased the apoptosis resistance of HG-induced senescent RTECs with upregulated cleaved caspase 3 and increased the percentage of apoptotic RTECs. Mechanistically, DcR2 mediated apoptotic resistance in senescent RTECs by enhancing GRP78-caspase 7 interactions and promoting Akt phosphorylation. Thus, DcR2 mediated the apoptotic resistance of senescent RTECs and renal fibrosis by interacting with GRP78, indicating that targeting the DcR2-GRP78 axis represents a promising therapeutic strategy for DN.

Effects of intracavitary administration of elemene combined with nedaplatin on malignant pleural effusion.

AIM: To investigate the therapeutic effect of Elemene combined with Nedaplatin (ECN) on malignant pleural effusion (MPE) and its adverse reactions. METHOD: A retrospective study was conducted, three hundred and fifty-two patients with MPE were divided into two groups according to different treatment methods. One hundred and eighty-nine patients were given intrathoracic injection of ECN and classified in ECN group; one hundred and sixty-three cases in the Nedaplatin group were given intrathoracic injection of nedaplatin. Routine treatments were used to prevent adverse reactions. RESULT: The effective rate of the ECN group was 57.05%, and that of the Nedaplatin group was 23.08%. The comparison results of adverse reactions between the two groups showed that there was no significant difference in leukopenia, thrombopenia, anemia, vomitting and diarrhea, fever, hepatic damage and renal damage. The level of thoracalgia in the ECN group was higher than that in the Nedaplatin group. There was no significant change in the number of CD8+ T cells between the two groups after treatment. The number of CD4+T cells in the ECN group increased after treatment was higher than the Nedaplatin group after treatment. CONCLUSION: ECN treatment can improve clinical control of MPE with no serious adverse reaction, can effectively reduce the immunosuppressive effect of nedaplatin and enhance the immune function of MPE patients which is worthy of clinical application.

Tumor necrosis factor alpha gene polymorphism is associated with the outcome of trauma patients in Chinese Han population.

BACKGROUND: Tumor necrosis factor (TNF) is a proinflammatory cytokine that plays a major role in the sepsis and multiple organ dysfunction secondary to major trauma. The purpose of this article was to research the clinical relevance of the TNF gene polymorphism in patients with major trauma. METHODS: Three hundred six patients with major trauma were prospectively recruited. The TNF gene polymorphisms were genotyped using restriction fragment length polymorphism analysis. Plasma TNF-α levels were determined with enzyme-linked immunosorbent assay. Sepsis morbidity rate and multiple organ dysfunction scores were accessed. RESULTS: The TNF-α/-308 polymorphism was shown to be well associated with increased capacity of peripheral leukocytes to produce TNF-α in response to ex vivo lipopolysaccharide stimulation in trauma patients at admission. Results from association study indicated that trauma patients carrying the TNF-α/-308/A allele were more likely complicated with sepsis. CONCLUSIONS: The TNF-α/-308 polymorphism might be used as a biomarker for the assessment of outcome of trauma patients, but the TNF-ß/252 gene polymorphism might not influence the development of complications in patients with major trauma.

High-dose HOOK effect in urinary DcR2 assay in patients with chronic kidney disease.

BACKGROUND: Urinary DcR2 (uDcR2) is a biomarker for the early detection the tubulointerstitial injury (TII) in patients with chronic kidney disease (CKD), but the high-dose hook effect may lead to falsely low or even negative results when using an enzyme-linked immunosorbent assay (ELISA). This study aimed to investigate if the high-dose hook effect exists with ELISA testing, and to uncover a potential approach for reducing this effect. METHODS: 72 CKD patients were recruited and categorized into four groups based on TII scores. uDcR2 was measured in undiluted and serially diluted (two-, four-, eight- and 16-fold dilutions) urine using an ELISA kit. The results from the assay were normalized to urinary creatinine. We evaluated the correlation between uDcR2/cre levels at different dilutions and renal histological parameters. Receiver operating characteristic (ROC) curves were generated to examine the value of uDcR2/cre for predicting TII. RESULTS: uDcR2/cre levels in the undiluted urine were significantly higher in patients with CKD than those in the control. However, higher TII scores did not yield higher levels of uDcR2/cre in the undiluted urine. After serial dilution, uDcR2/cre levels were highest with the four-fold dilution. A positive correlation was found between uDcR2/cre levels at different dilutions and TII scores, with the highest correlation coefficient and the largest AUC being observed at the four-fold dilution. CONCLUSIONS: The high-dose hook effect was apparent during ELISA testing of uDcR2 in CKD patients, yet dilution of the urine samples neutralized this effect. However, the use of a four-fold dilution of urine for uDcR2/cre testing may eliminate the high-dose hook effect and make it possible to effectively monitor the severity of TII in CKD patients.

Isolation and identification of senescent renal tubular epithelial cells using immunomagnetic beads based on DcR2.

Cell senescence plays a major role in the progression of tumors and chronic conditions such as diabetes and chronic kidney disease. Senescent cells are an important model for the study of aging-related diseases, and there is currently no efficient method for sorting out senescent cells. Decoy receptor 2 (DcR2) is a transmembrane receptor of the tumor necrosis factor superfamily, which is specifically expressed in senescent cells. In this study, we used magnetic activated cell sorting (MACS) isolation of a highly-pure populations DcR2-positive renal tubular epithelial cells (RTECs) based on three senescent cell models including the fifth passage cells, advanced glycation end-products (AGEs)- and H2O2-induced cells. The percentages of DcR2 positive RTECs in G1 and S phases increased by 20% and 4%, respectively, as compared to that in the pre-sorted cells. The positivity rates of SA-ß-gal, p16, and senescence-associated heterochromatin foci (SAHF) in DcR2-positive RTECs were about 40%, 30%, and 44% higher than that prior to cell sorting. The levels of IL-6 and TGF-ß1 in the supernatant were increased by 1.7 and 1.5 folds, respectively, as compared to that observed prior to sorting. No significant cell death was observed after 5days of continuous culture. Ki-67 positive expression rate in DcR2 negative RTECs was significantly higher than that in DcR2 positive RTECs after MACS. We demonstrated the use of DcR2 to classify live, senescent RTECs with a high specificity and stability. Our findings lay the foundation for further study of senescent RTECs in the progression of chronic kidney disease.

YAP is closely correlated with castration-resistant prostate cancer, and downregulation of YAP reduces proliferation and induces apoptosis of PC-3 cells.

Yes-associated protein 65 (YAP65) has been implicated as an oncogene, and its expression is increased in human cancer. Previous studies have demonstrated that alterations in YAP activity may result in tumourigenesis of the prostate. With androgen deprivation therapies becoming progressively ineffective, often leading to life­threatening androgen­resistant prostate cancer (CRPC). The present study aimed to analyse the role of YAP in prostate cancer (PCa), particularly in CRPC. YAP protein was detected using immunohistochemistry and western blot analysis in different prostatic tissues. In addition, three specific RNA interference vectors targeting the human YAP gene were synthesised, and PC­3 cells with a stable inhibition of YAP were obtained by transfection. MTT, flow cytometry, reverse transcription­quantitative polymerase chain reaction and western blot assays were used to analyse the effects of YAP inhibition on the proliferation and apoptosis of PC­3 cells. The frequency of cells that were positive for YAP protein in PCa (78.13%) was significantly higher, compared with para­PCa (26.67%; P=0.007) and benign prostatic hyperplasia (0%; P=0.002). The frequency of cells, which were positive for the expression of YAP exhibited a positive correlation (P=0.008) with the Gleason score, the tumour­node­metastasis staging (P=0.033) and the level of prostate specific antigens (P=0.0032) in PCa. The proliferative capacity of the transfected group was significantly lower, compared with the negative control group (P=0.022). The cell­cycle of the transfected group was arrested in the G1 stage, which was detected using flow cytometry, and there was a significant increase in the apoptosis of cells in the transfected group (P=0.002). The mRNA and protein levels of TEA domain family member 1 were inhibited in the transfected group (P=0.001 and P=0.00, respectively). Therefore, it was concluded that gene transcription and protein expression of YAP may be involved in the development of PCa, particularly CRPC, and may be a novel biomarker for investigation of the occurrence and progression of CRPC. However, the mechanism underlying the modulation of YAP in CRPC remains to be fully elucidated.

Methylation­associated inactivation of LATS1 and its effect on demethylation or overexpression on YAP and cell biological function in human renal cell carcinoma.

Large tumor suppressor 1 (LATS1) gene is one of the key factors in Hippo signaling pathway. Inactivation of LATS1 by promoter methylation was found in colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), astrocytoma, breast cancer and it was proved to be a tumor suppressor. However, its role is unclear in renal cell carcinoma (RCC). In this study, the expression of LATS1 was determined by reverse transcription polymerase chain reaction (RT­PCR) and immunohistochemistry in 30 pairs of RCC tissues and matched normal kidney tissues and RCC cells. We found that the expression of LATS1 was markedly reduced in RCC tissues and cells, in the RCC tissue in 46.7% (14/30), while in the normal kidney tissues in 76.7% (23/30), and was associated with pathological grade and clinical stage of RCC. We detected methylation status of LATS1 by bisulfite sequence-PCR (BSP) in renal cancer cell line 786-O which lowers expression of LATS1, and we found it hypermethy-lated (in 97.5%). In addition, pharmacological demethylation using 5-Aza-2'-deoxycytidine (5-Aza) restored the expression of LATS1 mRNA and protein in 786-O cells, both LATS1 demethylation and overexpression of LATS1 downregulated the expression of Yes-associated protein (YAP), inhibited cell proliferation, induced cell apoptosis and cell cycle G1 arrest in 786-O cells. Thus, this report for the first time demonstrates the inactivation of LATS1 by promoter methy-lation and it is a tumor suppressor in kidney cancer. LATS1 may serve as a biomarker for possible early diagnosis and as a potential therapeutic target for human RCC.

YAP is overexpressed in clear cell renal cell carcinoma and its knockdown reduces cell proliferation and induces cell cycle arrest and apoptosis.

Yes-associated protein (YAP) has been reported to be an oncogene in a number of malignancies. It constitutes an important regulatory mechanism for the Hippo pathway, a key regulator of cell growth and apoptosis. The present study aimed to investigate the clinical significance and the role of YAP in the development of clear cell renal cell carcinoma (ccRCC). YAP expression levels were compared between ccRCC and adjacent normal renal tissues by RT-PCR and immunohistochemistry, respectively. YAP expression levels were then detected in ccRCC cell lines 786-0 and ACHN, as well as in human embryonic kidney 293 cells (HEK-293) using western blotting. Three specific YAP-shRNA lentiviral vectors were constructed and transfected into 786-0 cells, and then the mRNA and protein levels of YAP and downstream transcription factor TEAD1 were detected. Finally, the effects of YAP silencing on proliferation and the cell cycle distribution of 786-0 cells were detected by Cell Counting Kit-8 (CCK-8) and flow cytometry (FCM), respectively. The apoptosis rate was also analyzed by FCM. It was observed that the expression levels of YAP mRNA and protein in ccRCC tissues were higher than these levels in the adjacent normal renal tissues. The expression of YAP protein in ccRCC tissues was significantly correlated with clinical stage and differentiation. The YAP protein levels in the two ccRCC cell lines 786-0 and ACHN were significantly higher than that in the HEK-293 cells. Additionally, treatment of 786-0 cells with YAP-shRNA lentiviral vectors significantly reduced the expression levels of YAP and TEAD1 mRNA and protein. Further analyses in 786-0 cells in which YAP was decreased, revealed that cell proliferation was inhibited, cell cycle was arrested at the G1 phase and apoptosis was increased. These results indicate that YAP is an underlying oncogene in ccRCC and it may be a promising biomarker and therapeutic target of ccRCC.

Isolation and enrichment of PC-3 prostate cancer stem-like cells using MACS and serum-free medium.

Prostate cancer stem-like cells (PCSLCs) are considered to be the 'seed' of prostate cancer. The aim of this study was to confirm that the PC-3 cells, which we isolated and enriched from PC-3 cells through magnetic bead cell sorting (MACS) and serum-free medium (SFM) culture, were PCSLCs. Combinations of MACS, flow cytometry (FCM), SFM and immunocytochemistry (ICC) were used to ensure the positive expression of CD133 and CD44 on PC-3 and sphere-forming cell membranes. Self-renewal, multi-potential differentiation, unlimited proliferation and permanency assays were also applied to indentify whether the PC-3 cells exhibited the characteristics of cancer stem cells (CSCs). As a result, there was a low proportion of PCSLCs in the PC-3 cells. In the FCM assay, the proportion of cells expressing CD133 or CD44 in the PC-3 cells was 0.51 and 0.31%, respectively. In addition, we found that the proportion of PC-3 cells sorted by MACS that expressed CD133 was significantly increased compared with that of the sphere-forming cells cultured in SFM (99.09 vs. 84.80%, P<0.05), while no difference was observed in the proportion of cells expressing CD44 between them (99.88 vs. 99.82%, P>0.05). The expression of PAP and AR as detected by western blot analysis of induced PCSLCs was significantly increased compared with that of uninduced PCSLCs (P<0.05); the proliferation capacity of PCSLCs was significantly higher than that of both the PC-3 cells (P<0.05) and induced PCSLCs (P<0.05). Furthermore, the PCSLCs that were isolated from SFM and MACS both demonstrated certain characteristics of stem cells and should be considered as stem cell-like. These data may hold potential for further exploring the role of PCSLCs.

Rapid genotyping of three polymorphisms in the LBP gene using a triplex pyrosequencing approach.

We described a triplex polymerase chain reaction (PCR) and triplex pyrosequencing assay which allowed a simultaneous determination of three tag single nuleotide polymorphisms (tag SNPs) in the lipopolysaccharide-binding protein (LBP) gene: rs1780623, rs11536972 and rs2232618. This method enables a fast and cost-effective genotyping and a simultaneous determination of the three tag SNPs.

Clinical relevance of single nucleotide polymorphisms of the high mobility group box 1 protein gene in patients with major trauma in southwest China.

BACKGROUND: High-mobility group box protein 1 (HMGB1) is a pivotal late mediator involved in the development of sepsis and multiple organ dysfunction syndrome (MODS) in critically ill patients. While several single nucleotide polymorphisms (SNPs) have been demonstrated to be critical determinants for outcome of critically ill patients, little is known about the clinical relevance of SNPs of the HMGB1 gene up to date. METHODS: A total of 3 tag SNPs of the HMGB1 gene were selected using HapMap database and linkage disequilibrium analysis. The tag SNPs were genotyped using a pyrosequencing methodology in 556 unrelated patients with major trauma. Peripheral whole blood samples obtained immediately after admission were determined for HMGB1 production in response to ex vivo lipopolysaccharide (LPS) stimulation. RESULTS: The rs2249825 SNP and the haplotype TCG were significantly associated with LPS-induced HMGB1 production by peripheral blood leukocytes. There were also significant differences in sepsis morbidity rate and MOD scores among patients with different genotypes of the rs2249825. In addition, the patients with the wild-type haplotype TCG had a lesser sepsis morbidity rate and MOD scores than those without the TCG haplotype. CONCLUSION: A total of 3 SNPs might act as tag SNPs for the entire HMGB1 gene. The rs2249825 and the haplotype TCG might be used as relevant risk estimate for the development of sepsis and MODS in patients with major trauma.

Identification of haplotype tag SNPs within the entire TLR2 gene and their clinical relevance in patients with major trauma.

Toll-like receptor 2 (TLR2) signaling plays a critical role in orchestrating the innate immune response and the development of sepsis and subsequent organ dysfunction after trauma. The objectives of this prospective study were to identify haplotype tag single-nucleotide polymorphisms (htSNPs) within the entire TLR2 gene and to investigate their clinical relevance in patients with major trauma. A total of 410 patients with major trauma were prospectively recruited. The htSNPs of the TLR2 gene was determined using HapMap database and linkage disequilibrium analysis. The htSNPs were genotyped using polymerase chain reaction-restriction fragment length polymorphism method. The whole peripheral blood samples obtained immediately after admission were stimulated with bacterial lipoprotein and then determined for production of tumor necrosis factor-α, interleukin 8, and interleukin 10. Sepsis morbidity rate and multiple organ dysfunction (MOD) scores were accessed. Three SNPs (rs1898830, rs3804099, and rs7656411) were identified as htSNPs for the TLR2 gene. All of them were shown to be high-frequency SNPs in this study cohort. Two of them (rs1898830 and rs3804099) and the haplotype ATT were significantly associated with cytokine production by peripheral blood leukocytes in response to bacterial lipoprotein stimulation. Only rs3804099, however, was significantly associated with higher sepsis morbidity rate and MOD scores in patients with major trauma. In addition, the patients with the haplotype ATT had lower sepsis morbidity rate than those without the haplotype ATT. Therefore, three SNPs might act as htSNPs for the entire TLR2 gene in the Chinese population. The rs3804099 and the haplotype ATT might be used as relevant risk estimates for the development of sepsis and MOD in patients with major trauma.