Urinary exosomal mRNAs as biomarkers for predicting the therapeutic effect of renin-angiotensin system inhibitors in IgA nephropathy patients.

BACKGROUND: Renin-angiotensin system inhibitors (RASi) treatment is the basic therapy for IgA nephropathy (IgAN) patients. However, there is few of biomarker that can predict the efficacy of RASi. This study aimed to find urinary exosomal mRNAs related to the therapeutic effect of RASi in the treatment of proteinuria in IgAN patients. METHODS: We divided IgAN patients in screening cohort into A1 (proteinuria increase at 3 months), B1 (proteinuria decrease less than 50 % at 3 months), C1 (proteinuria decrease more than 50 % at 3 months) groups according to changes of proteinuria after treatment. The urinary exosomes were collected before biopsy, RNAs were extracted and analyzed with the microarray assay. The candidate genes were screened by differentially expressed genes (DEGs) analysis and then validated by quantitative real-time polymerase chain reaction (qPCR) in a validation cohort. A receiver operating characteristic (ROC) curve was used to evaluate gene performance in predicting therapeutic effect on RASi reducing proteinuria in IgAN patients. RESULTS: ECE1 and PDE1A mRNAs were significantly different among the three groups, and were gradually decreased among A1, B1 and C1 groups. In the validation cohort, the level of urinary exosomal ECE1 and PDE1A mRNAs were also significantly lower in A2 group compared with C2 group(ECE1, P < 0.001;PDE1A, P < 0.01). Besides, the level of ECE1 mRNA was also lower in B2 group compared with C2 group (P < 0.01). The ROC curve verified that urinary exosomal ECE1 and PDE1A gene level predicted RASi efficacy in IgAN patients with area under curve (AUC) 0.68 and 0.63 respectively. CONCLUSION: Urinary exosomal ECE1 and PDE1A mRNAs expression can serve as potential biomarkers for predicting the RASi efficacy to reduce proteinuria in IgAN patients.

Ultrasensitive DNA-Biomacromolecule Sensor for the Detection Application of Clinical Cancer Samples.

Diagnostic testing of biological macromolecules is of great significance for early warning of disease and cancer. Nevertheless, restricted by limited surface area and large steric hindrance, sensitive detection of macromolecules with interface-based sensing method remains challenging. Here, a "biphasic replacement" electrochemical aptamer-based (BRE-AB) sensing strategy which placed capture reaction of the biomacromolecule in a homogeneous solution phase and replaced with a small diameter of single-stranded DNA to attach to the interface is introduced. Using the BRE-AB sensor, the ultrasensitive detection of luteinizing hormone (LH) with the detection limit of 10 × 10-12 m is demonstrated. Molecular Dynamics simulations are utilized to explore the binding mechanism of aptamer and target LH. Moreover, it is confirmed that the BRE-AB sensor has excellent sensing performance in whole blood and undiluted plasma. Using the BRE-AB sensor, the LH concentrations in 40 clinical samples are successfully quantified and it is found that LH is higher expressed in breast cancer patients. Furthermore, the sensor enables simple, low-cost, and easy to regenerate and reuse, indicating potentially applicable for point-of-care biological macromolecules diagnostics.

Nonplanar Helicene Benzo[4]Helicenium for the Precise Treatment of Renal cell Carcinoma.

Immune and targeted therapy are becoming the first-line treatment for renal cell carcinoma (RCC). However, therapeutic outcomes are limited due to the low efficiency and side effect. Here, it is found that helicenes are able to exhibit an anticancer capability through changing the molecular structure from planar to nonplanar. Furthermore, the cytotoxicity in vitro and cancer inhibition ability of nonplanar helicenes increase with its aromatic rings' number. It is further demonstrated that benzo[4]helicenium shows the specific killing efficiency against the RCC cancer as compared to normal kidney cells. This is majorly originated from a more selective damage of benzo[4]helicenium for mitochondria and DNA in RCC cancer cells, not the normal kidney. The selective killing ability of benzo[4]helicenium makes it have potential to be used as a targeted drug for the precise treatment of RCC.

P2X7 receptor signaling promotes inflammation in renal parenchymal cells suffering from ischemia-reperfusion injury.

Extracellular adenosine triphosphate (ATP) and its receptor, P2X7 receptor (P2X7R), are playing an important role in the pathological process of renal ischemia-reperfusion injury, but their underlying mechanism remains unclear. Also, the effects of tubular epithelium-expressed P2X7 receptor on ischemia acute kidney injury is still unknown. The aim of this study is to clarify if this mechanism involves the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in the renal tubular epithelial cells. In our research, we used male C57BL/6 wild type and P2X7R (-/-) mice, cultured human proximal tubular epithelial cells, and kidneys from acute kidney injury patients. Mice underwent for unilateral nephrectomy combined with the lateral renal pedicle clamping. Cultured cells were subjected to hypoxia/reoxygenation or ATP. Apyrase and A438079 were used to block the extracellular ATP/P2X7 receptor pathway. We also constructed radiation-induced bone marrow (BM) chimeras by using P2X7R (-/-) mice and P2X7R (+/+) wild-type mice. P2X7 receptor deficiency protected from renal ischemia-reperfusion injury and attenuated the formation of NLRP3 inflammasome. By using BM chimeras, we found a partial reduction of serum creatinine and less histological impairment in group wild-type BM to P2X7R (-/-) recipient, compared with group wild-type BM to wild-type recipient. In renal tubular epithelial cells, hypoxia/reoxygenation induced ATP release and extracellular ATP depletion reduced the expression of active IL-1ß. ATP activated the NLRP3 inflammasome in renal tubular epithelial cells, which were blunted by transient silence of P2X7 receptor, as well as by P2X7 receptor blocking with A438079. In human samples, we found that patients with Stage 3 AKI had higher levels of P2X7 receptor expression than patients with Stage 1 or Stage 2. Extracellular ATP/P2X7 receptor axis blocking may protect renal tubular epithelial cells from ischemia-reperfusion injury through the regulation of NLRP3 inflammasome.

Differential toxicity mechanism of gold nanoparticles in HK-2 renal proximal tubular cells and 786-0 carcinoma cells.

Aim: To research the influence and mechanism of gold nanoparticles (AuNPs) with different size for HK-2 cells (kidney normal cells) and 786-0 cells (kidney cancer cells). Materials & methods: HK-2 cells and 786-0 cells were treated with 5 and 200 nm AuNPs at 1 and 10 µg/ml. The cell viability, intracellular reactive oxygen species levels, cell apoptosis, cell autophagy, and related cell signaling pathways were analyzed. Results: In HK-2 cells, AuNPs reduced the activity of Akt and mTOR and upregulated the expression of LC3 II. In 786-0 cells, the activity of p38 was upregulated, which leaded to the increase of caspase 3 and initiated apoptosis. Conclusion: AuNPs of 5 and 200 nm at 10 µg/ml exerted antitumor effect by prompting apoptosis and inhibiting proliferation, while autophagy was activated to protect HK-2 cells from AuNPs-induced cytotoxicity.

Programing Assembling/Releasing Multifunctional miRNA Nanomedicine to Treat Prostate Cancer.

MicroRNAs (miRNAs) therapy has shown to have great promise for the treatment of androgen-independent prostate cancer (AIPC) due to the low efficiency of hormonal therapy. However, instability of RNA and inefficiency of RNA therapy limit the use of miRNAs in the treatment of AIPC. Here, we report a pH/ATP-activated nanocomplexes for increasing cytosolic delivery of miR146a which can effectively inhibit the expression of epidermal growth factor receptor (EGFR) in AIPC. The nanocomplexes show identical suppressing effect in invasion, colony formation, migration ability, and growth of DU145 cells compared with Lipofectamine 2000 (lipo). But for in vivo experiments, the nanocomplexes vigorously suppress the growth of tumor volumes comparing to lipo group after five weeks' treatment. These results demonstrate the potential of the pH/ATP-activated nanocarriers for AIPC gene therapy.

Yes-associated protein regulates podocyte cell cycle re-entry and dedifferentiation in adriamycin-induced nephropathy.

Podocytes are terminally differentiated cells with little proliferative capacity. The high expression levels of cell cycle inhibitory proteins, including p21, p27, and p57, play an important role in maintaining the low level of proliferation of mature podocytes. In the present study, we aimed to explore the role of yes-associated protein (YAP) signalling in adriamycin-induced podocyte re-entry into the cell cycle and dedifferentiation. Proliferating cell nuclear antigen (PCNA)-, cyclin-dependent kinase 4 (CDK4)-, and Cyclin D1-positive podocytes were found in mice with adriamycin-induced nephropathy. In vitro, adriamycin administration increased the percentage of cells in S phase and the upregulation of mesenchymal-related marker proteins. CDK4 and cyclin D1 were significantly up-regulated after incubation with adriamycin. Overexpression of YAP in podocytes promoted their entry into the cell cycle; up-regulated cyclin D1, desmin, and snail2 expression and down-regulated Wilms' tumour 1 (WT1) and nephrin production. Recombinant murine FGF-basic induced podocytes to re-enter the cell cycle, inhibited WT1 and nephrin, and increased desmin and snail2 expression. Pretreating podocytes with verteporfin, an inhibitor of YAP/ TEA domain transcription factor (TEAD), decreased the adriamycin-induced overexpression of cyclin D1 and reduced the ratio of S-phase podocytes. This result was further verified by knocking down YAP expression using RNA interference. In conclusion, adriamycin induced podocytes to re-enter the cell cycle via upregulation of CDK4 and cyclin D1 expression, which was at least partly mediated by YAP signalling. Re-entry into the cell cycle induced the over-expression of mesenchymal markers in podocytes.

Serum miR-192 Is Related to Tubulointerstitial Lesion and Short-Term Disease Progression in IgA Nephropathy.

BACKGROUND/AIMS: Clinical manifestation and renal histology serve as the current "gold standard" for evaluation of renal lesions in IgA nephropathy. MiR-192 is regarded as a potential noninvasive biomarker for kidney disease. We sought to elucidate a relationship between intrarenal, serum, and urinary exosomal miR-192 with renal lesions and disease progression in IgA nephropathy. METHODS: Serum and urinary exosomal miR-192 were detected and correlated with clinical and pathological parameters from consecutive IgA nephropathy patients (n = 50) and healthy control patients (n = 25). Patients then received a follow-up of 24 months after biopsy. Disease progression was defined as a 40% reduction in estimated glomerular filtration rate. Expression of miR-192 was quantified by Taqman RT-PCR. Intrarenal -miR-192 was detected in 8 IgA nephropathy patients and 4 matched patients receiving kidney nephrectomy as controls via in situ hybridization. TGF-ß1 and E-cadherin expression in renal tissue was evaluated using immunohistochemistry. RESULTS: Intrarenal miR-192 was correlated with serum miR-192 (r = 0.690, p = 0.013). Both intrarenal and serum miR-192 decreased in IgA nephropathy patients and were correlated with estimated glomerular filtration ratio. Patients with lower intrarenal and serum miR-192 levels had a higher interstitial fibrosis and tubular atrophy score, more severe lesions in tubular atrophy, and interstitial inflammation. Renal E-cadherin expression was correlated (r = 0.484, p = 0.004) and TGF-ß1 was inversely correlated (r = -0.527, p < 0.001) with serum miR-192 in IgA. Patients with higher serum miR-192 had a lower disease progression rate over the course of 2 years (0 vs. 16%, p = 0.028). No correlation was found in urinary exosomal miR-192 with the clinical and pathological parameters mentioned earlier. CONCLUSIONS: Lower serum miR-192 in IgA nephropathy patients indicates lower intrarenal miR-192 expression, more severe tubular atrophy, interstitial inflammation, and fibrotic tendency (with higher TGF-ß and E-cadherin in renal miR-192). IgA nephropathy patients with higher serum miR-192 are less likely to have renal function decline over the course of 2 years.

Pharmacological inhibition of heparin-binding EGF-like growth factor promotes peritoneal angiogenesis in a peritoneal dialysis rat model.

BACKGROUND: Molecular mechanisms of peritoneal dialysis (PD) ultrafiltration failure, peritoneal neo-angiogenesis, and fibrosis remain to be determined. We aimed to determine the role of heparin-binding EGF-like growth factor (HB-EGF) inhibition on angiogenesis of peritoneal membrane in a PD rat model. METHODS: 32 male Wistar rats were assigned into (1) control group; (2) uremic non-PD group: subtotal nephrectomy-induced uremic rats without PD; (3) uremic rats subjected to PD: uremic rats that were dialyzed with Dianeal® for 4 weeks; (4) CRM 197 group: dialyzed uremic rats were supplemented with CRM197, a specific HB-EGF inhibitor. Peritoneal transport function was examined by peritoneal equilibration test. Expression of HB-EGF and EGFR in peritoneal samples were examined by real-time PCR, immunohistochemical staining, and western blot. RESULTS: Progressive angiogenesis and fibrosis were observed in uremic PD rats, and there were associated with decreased net ultrafiltration (nUF), increased permeability of peritoneal membrane, and reduced expression of HB-EGF and EGFR protein and mRNA in uremic PD rats compared to uremic non-PD or control groups (both p < 0.05). CRM197 significantly induced peritoneal membrane permeability, decreased nUF, increased higher vessel density, and reduced pericyte count compared to that of uremic PD rats. The levels of HB-EGF and EGFR expression negatively correlated with vessel density in peritoneal membrane (both p < 0.001). CONCLUSION: PD therapy was associated with peritoneal angiogenesis, functional deterioration, and downregulation of HB-EGF/EGFR. Pharmacological inhibition of HB-EGF promoted PD-induced peritoneal angiogenesis and fibrosis and ultrafiltration decline, suggesting that HB-EGF downregulation contributes to peritoneal functional deterioration in the uremic PD rat model.

Fe3O4 nanoparticles and cryoablation enhance ice crystal formation to improve the efficiency of killing breast cancer cells.

The key problem of cryoablation is that only freezing is often unable to kill the capillaries at tumor edges, leading to a high rate of recurrence. Here, we found that Fe3O4 nanoparticles were highly useful to improve the freezing capability of cryosurgery due to their ability to alter intracellular ice formation (IIF) and growth in tumor cells. The killing efficiency of cryoablation for MCF-7 breast cancer cells can be expected to be enhanced as the Fe3O4 nanoparticles concentration increased, it was mainly because that more IIF was induced by the participation of Fe3O4 nanoparticles during freezing, recrystallization and thawing. Furthermore, our results also showed that recrystallization contributed to the formation of extracellular embryonic crystals, which was capable of enhancing the efficiency of killing MCF-7 cells. This research is to develop an understanding of the mechanism of the cryoablation enhancing the killing efficiency in the presence of the Fe3O4 nanoparticles, and to promote their further application in tumor therapy.

Implications of dynamic changes in miR-192 expression in ischemic acute kidney injury.

PURPOSE: Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) with poor outcomes. While many important functions of microRNAs (miRNAs) have been identified in various diseases, few studies reported miRNAs in acute kidney IRI, especially the dynamic changes in their expression and their implications during disease progression. METHODS: The expression of miR-192, a specific kidney-enriched miRNA, was assessed in both the plasma and kidney of IRI rats at different time points after kidney injury and compared to renal function and kidney histological changes. The results were validated in the plasma of the selected patients with AKI after cardiac surgery compared with those matched patients without AKI. The performance characteristics of miR-192 were summarized using area under the receiver operator characteristic (ROC) curves (AUC-ROC). RESULTS: MiRNA profiling in plasma led to the identification of 42 differentially expressed miRNAs in the IRI group compared to the sham group. MiR-192 was kidney-enriched and chosen for further validation. Real-time PCR showed that miR-192 levels increased by fourfold in the plasma and decreased by about 40% in the kidney of IRI rats. Plasma miR-192 expression started increasing at 3 h and peaked at 12 h, while kidney miR-192 expression started decreasing at 6 h and remained at a low level for 7 days after reperfusion. Plasma miR-192 level in patients with AKI increased at the time of ICU admission, was stable for 2 h and decreased after 24 h. AUC-ROC was 0.673 (95% CI: 0.540-0.806, p = 0.014). CONCLUSIONS: Plasma miR-192 expression was induced in a time-dependent manner after IRI in rats and patients with AKI after cardiac surgery, comparably to the kidney injury development and recovery process, and may be useful for the detection of AKI.

Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells.

The application of Fe3O4 nanoparticles (NPs) has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mechanisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm). Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application.

Impact of thawing on reference gene expression stability in renal cell carcinoma samples.

More and more samples are obtained from biobanks for biomedical research; however, some of these samples may undergo thawing before processing. We aim to evaluate the reference gene expression stability in thawed renal cell carcinoma samples. Sixteen matched malignant and nonmalignant renal tissue samples were obtained and each sample was divided into 4 aliquots before being snap frozen and stored at -80°C. By quantitative real-time polymerase chain reaction, a time-course study was conducted on the thawed tissue to evaluate the expression stability of a panel of the 10 most frequently used reference genes in renal cell carcinom samples: ACTB, ALAS1, B2M, GAPDH, HMBS, HPRT, PPIA, RPLP0,TBP, and TUBB. As shown by geNorm M values, PPIA was the most stable gene at the 0-, 15-, and 30-minute time points (M=0.82, 0.85, and 0.76, respectively), whereas GAPDH was ranked last at the 5-, 15-, and 30-minute time points (M=1.38, 1.44, and 1.39, respectively). A positive correlation was found by linear regression between the thawing time and 2 to the power of crossing point values of all candidate reference genes (P<0.05). The mean coefficient of variance of all reference genes increased significantly at time points 5, 15, and 30 minutes compared with 0 minutes (P<0.01). In conclusion, using the geNorm algorithm, PPIA was identified as the most stably expressed gene between malignant and nonmalignant renal tissue samples that were thawed for similar time periods. All the reference genes showed high variations along with the thawing time; it should be recommended to use a combination of several candidate reference genes when comparing samples thawed for different time periods.

Urinary neutrophil gelatinase-associated lipocalin and L-type fatty acid binding protein as diagnostic markers of early acute kidney injury after liver transplantation.

OBJECTIVE: We examined the value of two potential novel urinary biomarkers, neutrophil gelatinase-associated lipocalin (NGAL) and L-type fatty acid binding protein (L-FABP), in diagnosing acute kidney injury (AKI) in liver transplant recipients. METHODS: NGAL and L-FABP in urinary sample from Twenty-five patients before surgery and at 2, 4, 6, 12, 24, 48, 72 and 120 h after the anhepatic phase were tested. Standard statistics were used along with receiver-operating characteristic (ROC) analysis to evaluate the diagnostic value of selected markers. RESULTS: Urinary NGAL was only slightly elevated at 2 h in the non-AKI group while rose and stayed high from 2-6 h in the AKI group. However, urinary L-FABP rose transiently in both groups 2-120 h following surgery. The level of urinary NGAL presented differences at 2-6 h (p < 0.05) and urinary L-FABP at 4 h (p < 0.05) between AKI and non-AKI groups. ROC analysis showed that area under the curves (AUCs) of NGAL were 0.766, 0.773, and 0.773 at 2, 4 and 6 h respectively while 0.760 of L-FABP at 4 h. CONCLUSION: Urinary NGAL rather than L-FABP appeared to be a sensitive and specific marker of AKI in liver transplant recipients.

Impact of warm ischemia on gene expression analysis in surgically removed biosamples.

Surgically removed samples of high quality provide more accurate and reliable results in downstream molecular assays. Some factors, including the type of anesthesia, surgical manipulation, transport time and mode, preservation method, storage length, and number of freeze-thaw cycles, can affect biosample quality and the subsequent gene expression analysis. Warm ischemia resulting from these factors has a substantial effect on biosample quality and is the focus of this mini review. We classified the effects of warm ischemia on gene expression as (i) warm ischemia-induced metabolic activity (WIMA) in living cells and (ii) warm ischemia-induced RNA degradation (WIRD). The differential effects of WIMA and WIRD on gene expression analysis appear to depend on the period after surgical removal. WIMA predominantly affects gene expression during the early stage after surgery, whereas WIRD has a more significant effect after tissue thawing. By a literature review, we also found that RNA isolated from surgically removed biopsies is stable, and high-quality RNA can be obtained for most nonfixed human tissue maintained at room temperature during the early period after surgery. Understanding these characteristics of gene expression variation should help biomedical researchers to avoid misleading gene expression results.

Attitudes towards biosample donation in andrology patients.

Clinical left-over biosamples are an important source for medical research. Our aim is to ascertain the attitudes of andrology patients towards biosample donation. A survey of 866 andrology patients with diverse andrology conditions was conducted in Shanghai, China from May 2010 to December 2010. The response rate was 75.8%. Of the study patients, 61.4% (95% confidence interval, CI: 0.58-0.65) voiced the opinion that they were willing to donate the residual biosample; 32.0% refused to donate; and 6.6% stated that they were undecided. Unwillingness to give sample was independently associated with having a low level of education (p = 0.047, OR = 0.63, 95% CI: 0.39-0.995) and being infertile patients (p = 0.03, OR = 0.61, 95% CI: 0.39-0.95), while willingness was significantly associated with being cancer patients (p = 0.04, OR = 1.90, 95% CI: 1.03-3.50), being aged 18-29 years (p < 0.01, OR = 5.88, 95% CI: 2.85-12.16) and being aged 30-44 years (p = 0.01, OR = 2.17, 95% CI: 1.20-3.90). Most andrology patients would want to authorize every future research by themselves (82.4%) and obtain their individual research results (75.7%). Less than half of the willing respondents would want to donate semen (44.1%) and testis (41.3%). The results suggested that andrology patients' willingness to donate samples was low, especially in case of infertile patients. More studies are required to elucidate further causes for the low willingness to donate sample among andrology patients.

Functional metabotropic glutamate receptors 1 and 5 are expressed in murine podocytes.

In non-neuronal cells, glutamate is an extracellular signaling mediator. Since podocytes have glutamate-containing vesicles, we sought to determine glutamate receptor presence and action in glomerular cells. The metabotropic glutamate receptors (mGluR) 1, 5, 6, and 8 were found to be expressed in mouse brain and glomeruli; predominantly in podocytes. In two models of proteinuria (BalB/C mice with puromycin aminonucleoside- and doxorubicin-induced podocyte injury) we found that the selective mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) attenuated albuminuria and improved the expression of the podocyte marker WT-1. TUNEL staining showed that the number of podocytes undergoing apoptosis was inversely correlated with the number of WT-1-positive cells in glomeruli. When podocytes were treated with DHPG in vitro, they generated cyclic AMP and activated CREB (cyclic AMP response element binding protein). The selective mGluR1/5 antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid, the adenylate cyclase inhibitor SQ22536, and RNA interference knockdown of mGluR1 or mGluR5 all prevented DHPG-induced cAMP generation and CREB activation. DHPG inhibited apoptosis and the decrease of aminonucleoside-induced mitochondrial membrane potential in podocytes but had no effect in the presence of SQ22536 with knockdown mGluR1 or mGluR5. Thus, functional mGluR1 and mGluR5 are expressed in podocytes and their activation protects against albuminuria and podocyte apoptosis, processes that are, at least in part, dependent on cAMP.

[Effect of ethyl pyruvate on expression of inflammatory factors and mitogen-activated protein kinase proteins in renal ischemic/reperfusion injury in BABL/c mice].

OBJECTIVE: To investigate the effects of ethyl pyruvate (EP) on expression of proinflammatory related gene and proteins of mitogen-activated protein kinase (MAPK) in renal tissues in ischemic/reperfusion (I/R) injury in mice. METHODS: Fifty male BABL/c mice were randomly divided into sham operation group (n=8), model group (n=10), and EP treatment group (n=32). EP treatment group was subdivided into EP pretreatment group (administration of 40 mg/kg EP 30 minutes before reproduction of model, n=8), and 4, 6 and 12 hours treatment groups (administration of 40 mg/kg EP 4, 6 and 12 hours after reproduction of model, respectively, n=8 in each group). Bilateral renal artery was occluded with a microvascular clamp for 30 minutes to reproduce kidney I/R injury model, and the kidney was harvested at 24 hours after I/R. The mRNA expressions of interleukins (IL-1ß, IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and high-mobility group box 1 (HMGB1) were determined by real time reverse transcription-polymerase chain reaction (RT-PCR). The changes in protein levels of MAPKs [extracellular regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38MAPK] were determined by Western blotting analysis. RESULTS: Real-time PCR assay showed that the mRNA expressions of IL-1ß, IL-6, TNF-α, ICAM-1, HMGB1 in renal tissue were much higher than those in sham operation group (IL-1ß: 12.05±8.08 vs. 3.18±1.13, IL-6: 10.26±6.85 vs. 0.81±0.34, TNF-α: 5.83±3.85 vs. 0.67±0.34, ICAM-1: 3.87±2.02 vs. 0.29±0.13, HMGB1: 652.82±78.50 vs. 112.31±32.50, all P<0.05); and the expression in EP treatment groups was markedly down-regulated than that in model group, especially in 12-hour treatment group (0.45±0.26, 0.66±0.13, 0.21±0.11, 0.05±0.02, 212.26±3.20, respectively, all P<0.05). Western blotting analysis revealed that the expression of the phosphorylated forms of ERK1/2, JNK, p38MAPK proteins was significantly higher than in sham operation group (p-ERK1/2: 1.13±0.38 vs. 0.48±0.34, p-JNK: 1.40±0.15 vs. 0.36±0.15, p-p38MAPK: 0.47±0.15 vs. 0.21±0.17, all P<0.05); the expression of the phosphorylated forms of ERK1/2, JNK, p38MAPK in each EP treatment group was significantly down-regulated compared with that in model group (all P<0.05). CONCLUSION: EP can effectively protect kidney from acute injury produced by I/R, which may be related to the regulation of proinflammatory genes and the MAPKs in renal tissue.

[Icodextrin improve angiogenesis of peritoneal membrane in continuous ambulatory peritoneal dialysis patients].

OBJECTIVE: To observe the effect of icodextrin on peritoneal membrane angiogenesis in continuous ambulatory peritoneal dialysis (CAPD) patients. METHODS: This was a randomized double-blind perspective study of CAPD patients at our center between January 2006 to December 2006. The patients were randomized to receive either 7.5% icodextrin (ICO, n = 27) or glucose (GLU, n = 27) solution at night for 4 weeks. Peritoneal membrane function was defined as dialysate dwell for 4 hours to plasma ratio of creatinine (4 h D/Pcr) at baseline. Ultrafiltration volume, creatinine clearance (Ccr), VEGF and IL-6 in peritoneal effluent during the long night dwell (UF) dialysate were measured at baseline and after 4 weeks. The VEGF appearance was used to adjust the influences of dwell time and ultrafiltration volume. RESULTS: A total of 54 patients were enrolled. The baseline conditions showed no difference between the groups. After 2 and 4 weeks of therapy, both net UF and peritoneal creatinine clearance of long dwell were significantly higher in the ICO group than the GLU group. VEGF in night dwell PD solution was positively correlated with D/PCr (r = 0.68, P < 0.01)and negatively correlated to 4 hour ultrafiltration volume (r = -0.51, P < 0.01). The VEGF appearance was comparable between two groups at baseline. After a follow-up of 4 weeks, the VEGF appearance had an increasing tendency in the GLU group and a decreasing tendency in the ICO group but there was no significant difference. The ΔVEGF appearance (VEGF appearance in 4 week-VEGF appearance at baseline) was different between the GLU and ICO groups (9.5 ± 20.2 vs -13.4 ± 26.1, P < 0.01). IL-6 in night dwell dialysate had no difference between two groups. CONCLUSION: As compared with glucose-based solution, 7.5% icodextrin significantly decreases the local VEGF level in dialysate.

Clinical usefulness of novel biomarkers for the detection of acute kidney injury following elective cardiac surgery.

BACKGROUND/AIMS: Acute kidney injury (AKI) is common following cardiac surgery and predicts a poor outcome. However, the early detection of AKI has proved elusive and most cases are diagnosed only following a significant rise in serum creatinine (SCr). We compared a panel of early biomarkers of AKI for the detection of AKI in patients undergoing heart surgery. This study included serum cystatin C (CyC) and urinary levels of neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), retinol-binding protein (RBP) and N-acetyl-beta-D-glucosaminidase (NAG). METHODS: We retrospectively identified 15 patients undergoing open cardiac surgery who developed AKI within 72 h postoperatively. For these, we identified 15 matched controls also having undergone surgery but without AKI. Serial serum and urine samples had prospectively been postoperatively obtained from all patients at 0, 2, 4, 6, 10, 24, 48 and 72 h after admission to the intensive care unit. AKI was defined as a >50% increase in SCr. CyC was measured by nephelometry, while NGAL, IL-18, and RBP were measured by ELISA and NAG was measured by spectrophotometry. The urinary biomarkers were normalized to urinary creatinine (UCr) concentration. Each marker was assessed at each time point for its predictive value using receiver operating characteristic curves to predict AKI. RESULTS: Following the exclusion of 1 case due to a urinary tract infection, the final cohort consisted of 29 patients aged 62.9 +/- 13.7 years with baseline SCr of 73.2 +/- 11.9 micromol/l. While there were no differences in the demographics between cases and controls, the aortic clamp time was predictably higher in AKI cases than in controls (60.6 +/- 13.9 vs. 43.0 +/- 9.2 min, p < 0.05). Each biomarker differed significantly between cases and controls for at least one time point. The optimal area under the curve (AUC) was for CyC at 10 h (sensitivity 0.71, specificity 0.92, cutoff 1.31 mg/l), NGAL at 0 h (sensitivity 0.84, specificity 0.80, cutoff 49.15 microg/g UCr), IL-18 at 2 h (sensitivity 0.85, specificity 0.73, cutoff 285.65 ng/g UCr), RBP at 0 h (sensitivity 0.75, specificity 0.67, cutoff 2,934.65 microg/g UCr) and NAG at 4 h (sensitivity 0.86, specificity 0.67, cutoff 37.05 U/mg UCr). Using a combination of all 5 biomarkers analyzed at the optimal time point as above, we were able to obtain an AUC of 0.98 (0.93-1.02, p < 0.001) in this limited sample. CONCLUSION: The use of serum and urinary biomarkers for the prediction of AKI in patients undergoing cardiac surgery is highly dependent on the sampling time. Of the evaluated markers urinary NGAL had the best predictive profile. The previously unstudied marker of urinary RBP showed similar predictive power as more established markers. By combining all 5 studied biomarkers we were able to predict significantly more cases, suggesting that the use of more than one marker may be beneficial clinically.