细胞培养、IR模型建立

201602 3D spheroid culture enhances survival and therapeutic capacities of MSCs injected into ischemic kidney.

• In vitro studies indicated that 3D spheroids of MSCs produced higher levels of extracellular matrix proteins (including collagen I, fibronectin and laminin), and exhibited stronger anti-apoptotic and anti-oxidative capacities than two-dimensional (2D) cultured cells
• Furthermore, 3D culture increased the paracrine secretion of cytokines by MSCs, including angiogenic factors (VEGF and basic fibroblast growth factor), anti-apoptotic factors (epidermal growth factor and hepatocyte growth factor), the anti-oxidative factor insulin-like growth factor and the anti-inflammatory protein tumour necrosis factor-alpha stimulated gene/protein 6.
• 3D spheroids of MSCs showed enhanced survival and paracrine effects in vivo.
• 3D spheroid culture enhances survival and therapeutic capacities of MSCs injected into ischemic kidney.
• when injected into the kidney of model rats
  with ischemia-reperfusion(I/R)-induced AKI, 3D spheroids were more beneficial in protecting the I/R kidney against apoptosis, reducing tissue damage, promoting vascularization and ameliorating renal function compared with 2D cultured cells.
• Therefore, the 3D culture strategy improved the therapeutic effects of MSCs, and might be promising for AKI treatment

201507 Dioclea violacea lectin ameliorates oxidative stress and renal dysfunction in an experimental model of acute kidney injury

• Treatment with Dvl lectin was performed prior to the induction of AKI. The animals were anesthetized with ketamine/xylazine (91.0/9.1 mg/kg, i.p.), placed on a heated surgical table, and either Dvl lectin (1 mg/kg, i.v.) or vehicle (VH; 100 µL of 0.9% NaCl, i.v.) was administered in penile vein.

• Thirty minutes after treatment, rats were submitted to renal ischemia reperfusion surgery. Briefly, a midline abdominal incision was made, and the blood supply to the kidneys was interrupted for 45 min by clamping the renal pedicles of both kidneys with a suture line (endocort, nº0, Laboratório Bruneau S.A.).

• The suture line was then released, and the kidneys were reperfused during 24 hours.

• In sham surgery, kidneys were exposed, but not clamped. Postoperative dehydration was prevented by subcutaneous administration of 1.0 mL of 0.9% NaCl

• Twenty-four hours after kidney reperfusion, renal function was determined using inulin and sodium para-aminohippurate (PAH) clearances to estimate glomerular filtration rate (GFR) and renal plasma flow (RPF), respectively.
  在肾脏受伤24小时后，使用菊粉和钠 对氨马尿酸的清除率来分别估计 肾小球滤过率和肾血浆流量

• The animals were anesthetized with sodium thiopental (50 mg/kg, i.p.), and a tracheostomy was performed to facilitate breathing, followed by insertion of polyethylene catheters into the femoral artery for collection of blood samples and into the femoral vein for the infusion of saline containing 3% mannitol. A catheter was inserted into the bladder for urine collection. The arterial catheter was connected to a pressure transducer (Cobe Laboratories, USA), which was plugged into a pressure-processor amplifier and data acquisition system (MP100, Biopac Systems, USA) for continuous monitoring of arterial pressure and heart rate.

• A 30 min infusion with saline containing 3% mannitol at a rate of 0.06 ml/min was performed, followed by a priming injection of 1 ml of saline containing inulin (300 mg/kg) and PAH (6.66 mg/kg). After that, an infusion at the rate of 0.1 ml/min containing saline, inulin (15 mg/ml), PAH (4 mg/ml), and 3% mannitol was started. Four urine and blood samples were collected at 30 min intervals, starting 30 min after injection of the priming solution for a total of 4 samples. Hematocrit was measured using a heparinized capillary tube. Plasma and urinary inulin and PAH concentrations were measured using a colorimetric assay [[21]. Blood samples were also used for plasma urea quantification through spectrophotometry.
  Renal blood flow (RBF) and renal vascular resistance (RVR) were determined as previously described [[22
  ]. Briefly, RBF was calculated by the equation RBF=RPF/(1-hematocrit), and RVR was calculated using the equation RVR=MAP/RBF.

• Myeloperoxidase assay 髓过氧物酶分析
  Myeloperoxidase (MPO) activity was measured as an index of inflammation, as described previously [[24,[25]. One hundred mg of kidney was homogenized in 2000 µL of potassium phosphate buffer 0.05 M containing 5 mg/mL of hexadecyl trimethyl ammonium bromide and 1.85 mg/mL of ethylenediaminetetraacetic acid (pH 6.0), using a homogenizer. The homogenate was centrifuged at 12500 g for 15 min at 4°C to obtain the supernatant, which was used to measure MPO activity. Seventy-five microL of supernatant was mixed with 1425 µL of phosphate buffer containing 0.167 mg/mL of o-dianisidine hydrochloride and 0.05% hydrogen peroxide (H2O2). The change in absorbance was measured at 460 nm for 10 min. Results are expressed as units of MPO activity per gram of tissue, where 1 unit is the quantity of enzyme able to convert 1 mM of H2O2
  to water in 1 min at 25°C.

• Cell samples 细胞采样
  Enriched kidney cell fractions from different groups were prepared as standardized in our laboratory based on previous studies [[26,[27]. The kidney was divided into cortex and medulla, grossly triturated with surgical scissors, and incubated with an extraction solution containing trypsin (Sigma-Aldrich, St. Louis, MO, USA) to dissociate the cells. The cell extract was filtered through a nylon screen (BD Falcon 70 µm) to remove cell debris. The samples were washed twice in phosphate-buffered saline (PBS) and stored at -80°C until further analysis.
  皮质和髓质分开捣碎

• NO intracellular detection 细胞内NO检测
  The measurement of nitric oxide (NO) was performed as previously described . Briefly, a NO-sensitive fluorescent probe, 4,5-diaminofluorescein-2/diacetate (DAF-2/DA: 2 μM), was added to the cell suspension (106cells) and incubated at 37°C for 180 minutes in the dark. For positive control, samples were treated with 100 μM sodium nitroprusside. Cells were then washed, resuspended in PBS and immediately analyzed by flow cytometry (FACSCanto II, Becton Dickinson, CA). Data were analyzed using the FACSDiva software (Becton Dickinson), and overlay histograms were constructed using FCS Express software (De Novo). For quantification of DAF fluorescence, samples were acquired in duplicate, and 10,000 events were used for each measurement. Cells were excited at 488 nm, and fluorescence was detected using a 530/30 band-pass filter. Data were expressed as geometric mean fluorescence intensity.

• ROS detection 活性氧
  Reactive oxygen species (ROS) analysis was performed by flow cytometry as previously described. Dihydroethidium (DHE) and 2’,7’-dichlorofluorescein diacetate (DCF-DA) were used to detect intracellular superoxide anion (•O2-) and hydrogen peroxide (H2O2), respectively. By its ability to freely permeate cell membranes, DHE is extensively used to monitor •O2-
  production. Upon reaction with •O2-, DHE is rapidly oxidized forming ethidium, a red fluorescent product that intercalates with DNA and amplifies the red fluorescence signal. DCF-DA is a cell permeant indicator for H2O2 production. It is nonfluorescent until oxidation occurs within the cell, converting it to the fluorescent form that remains trapped in the cell. DHE (160 mM) and DCF-DA (20 mM) were added to the cell suspension (106 cells) and incubated at 37°C for 30 min, in the dark, to estimate intracellular •O2-or H2O2 concentration. For positive control, samples were treated for 5 min with 10 μM doxorubicin or 50 mM H2O2 to create oxidative stress, but without toxicity to the cells, whereas for negative control, the cells were incubated with ethanol. Cells were then washed, resuspended in PBS, and kept on icefor immediate detection by flow cytometry (FACSCanto II, Becton Dickinson, CA). Data were analyzed using the FACSDiva software (Becton Dickinson), and overlay histograms were constructed using FCS Express software. For quantification of DHE and DCF fluorescence, samples were acquired in duplicate, and 10,000 events were used for each measurement. Cells were excited at 488 nm, and DHE and DCF fluorescence were detected using, respectively, 585/42 and 530/30 band-pass filters. Data were expressed as geometric mean fluorescence intensity.

• hROS detection

• Apoptosis 凋亡