Objective: To investigate the effect and mechanism of atorvastatin (ATV) on the inflammatory response of human renal tubular epithelial cells (HK-2 cells) induced by calcium oxalate crystals. Methods: HK-2 cells were divided into control group (normal medium), ATV group (after 3 h pretreatment with 40 μmol/L ATV, replaced with normal medium), calcium oxalate crystal stimulation group (4 mmol/L calcium oxalate crystal) and ATV treatment group (after 3 h pretreatment with 40 μmol/L ATV, replaced with 4 mmol/L calcium oxalate crystals). After 12 h, the cells were collected, and the expression levels of NLRP3 and Cleaved caspase-1 were detected by immunohistochemical staining and Western blotting. The expression level of NF-κB was detected by immunofluorescence and Western blotting. The cell culture supernatant was collected to detecte the concentrations of interleukin-1β (IL-1β) and interleukin-18 (IL-18) by enzyme linked immunosorbent assay (ELISA). Results: Western blot analysis showed that the relative expression of NLRP3 (0.125±0.013 vs. 0.135±0.007) and Cleaved caspase-1 (0.090±0.014 vs. 0.095±0.006) was decreased in the ATV group compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NLRP3 (0.315±0.021 vs. 0.135±0.007, P<0.001) and Cleaved caspase-1 (0.235±0.008 vs. 0.095±0.006, P<0.001) was significantly increased in the calcium oxalate crystal stimulation group compared with the control group. While the relative expression of NLRP3 (0.245±0.007 vs. 0.315±0.021, P<0.05) and Cleaved caspase-1 (0.170±0.017 vs. 0.235±0.008, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The results of immunohistochemical staining showed that the expression trends of NLRP3 and Cleaved caspase-1 in each group were consistent with those obtained by Western blotting. The ELISA results showed that the concentration of inflammatory factors IL-1β [(162.00±21.21)pg/ml vs. (183.50±7.78) pg/ml, P>0.05] and IL-18 [(176.50±24.12)pg/ml vs.(182.50±20.51)pg/ml, P>0.05] in the ATV group was lower than that in the control group, but the difference were not statistically significant (P>0.05). The concentrations of IL-1β[(850.50±48.79)pg/ml vs. (183.50±7.78)pg/ml, P<0.001] and IL-18 [(526.00±39.61)pg/ml vs. (182.50±20.51)pg/ml, P<0.001] were significantly increased in the cell culture medium of the calcium oxalate crystal stimulation group compared with the control group, while the concentrations of IL-1β [(452.50±36.06)pg/ml vs. (850.50±48.79) pg/ml, P<0.01] and IL-18 [(403.50±23.33)pg/ml vs. (526.00±39.61)pg/ml, P<0.05] was significantly reduced in the cell culture medium of the ATV treatment group compared with the calcium oxalate crystal stimulation group. Western blot analysis showed that the relative expression of NF-κB (0.105±0.021 vs. 0.100±0.014) in the ATV group was decreased compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NF-κB (0.295±0.035 vs. 0.100±0.014, P<0.001) in the calcium oxalate crystal stimulation group was significantly increased compared with the control group. While the relative expression of NF-κB (0.160±0.012 vs. 0.295±0.035, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The expression of NF-κB by immunofluorescence staining was consistent with the results of Western blotting. Conclusions Calcium oxalate crystals can induce the inflammatory response of HK-2 cells, while ATV can exert anti-inflammatory effects by inhibiting the activation of NLRP3 inflammasome and decreasing the secretion of inflammatory factors IL-1β, IL-18 and the expression of NF-κB.

Calcium oxalate nephrolithiasis is the common disease of urinary surgery, its exact pathogenesis is still unclear.It is believed that the renal inflammatory injury induced by cell-crystal reaction plays an important role in the formation of intrarenal calcium oxalate crystals. Recent studies indicated that inflammation induced by cell-crystal reaction can cause renal cell damage, stimulate intracellular expression of NADPH oxidase, trigger the massive production of reactive oxygen species, activate nuclear factor-κB signaling pathway, release a large number of inflammatory factors, and cause inflammatory cascade effect of the kidney, thus promoting the accumulation, nucleation and growth of calcium salt crystals, eventually leading to the formation of intrarenal crystals and even stones. In this process, the regulatory factors and mechanisms involved include macrophages, NLRP3-high mobility group box-1 protein inflammation network, fetuin A, autophagy activation and other factors.

Objective To investigate the method for isolating and identification exosomes from the HK-2 cells exposed to high oxalate.Methods HK-2 cells were cultured to serum-free culture medium and treated with oxalate at the concentration from 0 mmol/L to 10.00 mmol/L for 48 h.CCK-8 assays were performed to measure the cells proliferation.Combined the cell morphology,observed under inverted microscope with statistical analysis,we finally 2.00 mmol/L oxalic acid as the experimental concentration.The HK-2 cell was exposed to 2.00 mmol/L oxalate for 48 h.The supernatants were collected.Exosomes were isolated and purified from the supernatants by ultracentrifugation.Transmission electron microscopy (TEM) was used to observe the morphology of isolated exosomes.Particle size of exosomes were detected with Nanosight technology.Western blot analysis was used to examine the experession of HSP70,CD63.Results CCK-8 assay showed that the cell viability in each group,including (100.0 ± 4.0) ％ in 0 mmol/L group;(97.7 ± 1.5)％ in the 0.25 mmol/L group;(97.3 ±2.1)％ in the 0.50 mmol/L group;(87.7 ± 2.1) ％ in the 1.00 mmol/L group;(76.0 ± 1.0) ％ in the 2.00 mmol/L group;(58.1 ± 2.6) ％ in the 4.00 mmol/L group;(52.7 ± 1.5) ％ in the 5.00 mmol/L group;(37.7 ± 3.2) ％ in the 8.00 mmol/L group;(31.3 ±2.0)％ in the 10.00 mmol/L group.The isolated exosomes demonstrated round or oval shape under TEM.The peak particle size was 56 nm,which the overall mean particle size was 87 nm.Those and particles with a diameter between 30-150 nm accounted for 91.2％.In this experiment,The expression of HSP70,CD63 could be detected in the isolated exosomes.However,only the expression of HSP70 could be detected in the HK-2 cells.Conclusions Under the treatment of 2 mmol/l oxalate for 48 hours,Ultracentrifugation can be used to isolate and purify exosomes efficiently from the HK-2 cells.This is helpful for further study of exosome as mediator of cell-to-cell communication.

AIM:To investigate the effects of osteogenic induction media and the medias containing different concentration of calcium on the induction of osteogenic differentiation of human renal fibroblasts in vitro.METHODS: Culturedhuman renal fibroblasts were divided into 5 groups in this experiment: osteogenic induction group (osteogenic inductionmedia), CaⅠgroup (0.5 mmol/L Ca2 + media), CaⅡgroup (1.5 mmol/L Ca2 + media), Ca Ⅲ group (2.5 mmol/LCa2 + media) and control group (PBS).The cell activity in each groups was measured by MTT assay .At 9th day, the cellcalcium Alizarin red S staining and alkaline phosphatase (ALP) Gomori calcium cobalt staining were performed respectivelyto observe the formation of calcium nidus and the expression of ALP .In addition, the expression of Runt-related transcriptionfactor 2 (Runx2) at mRNA and protein levels was determined by real -time PCR and Western blot, respectively.RE- SULTS: The remarkable positive signs which represented the formation of calcium nidus and the deposit of calcium objectsin all experiment groups were observed .The mRNA and protein expression of Runx2 in osteogenic induction group increasedin accordance with the induction time .Compared with control group, the mRNA and protein expression of Runx2 inthe CaⅠ ~Ⅲ groups increased gradually in a calcium concentration dependent manner at the 9th induction day.CON- CLUSION: Human renal interstitial fibroblasts show the potential activity in osteogenic differentiation induced by osteogen -ic induction media or high level calcium in vitro, which may be account for the cytological formation of the Randall ’splaque in the kidney.

BACKGROUND:More and more evidence suggests that macrophages and inflammation reactions are involved in the formation and development of nephrolithiasis. Previous studies have found that calculi crystals can stimulate macrophages to release high mobility group protein B1.
OBJECTIVE:To investigate the synergistic effect of high mobility group protein B1 in calcium phosphate induced release of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 from human macrophages.
METHODS:(1) The induced U937 cells were respectively stimulated with RPMI (blank), 100 mg/L calcium phosphate, 100μg/L high mobility group protein B1 and 100 mg/L calcium phosphate+100μg/L high mobility group protein B1 for 1, 2 and 4 hours to col ect cellsupernatant. (2) The induced U937 cells were respectively stimulated with 100 mg/L calcium phosphate, 100 mg/L calcium phosphate+10μg/L high mobility group protein B1, 100 mg/L calcium phosphate+50μg/L high mobility group protein B1, 100 mg/L calcium phosphate+100μg/L high mobility group protein B1 for 4 hours to col ect cellsupernatant. Levels of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 were determined by ELISA.
RESULTS AND CONCLUSION:The levels of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 in the cellculture supernatant of 100 mg/L calcium phosphate group and 100μg/L high mobility group protein B1 group were both higher than those in the blank group in a time-dependent manner (P<0.05). The levels of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 in the cellculture supernatant of different concentrations of high mobility group protein B1 groups were al higher than those in the 100 mg/L calcium phosphate group in a concentration-dependent manner (P<0.05). The results suggest that both calcium phosphate and high mobility group protein B1 can induce the release of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 from human macrophages and the high mobility group protein B1 has the synergistic effect with calcium phosphate to induce interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 from human macrophages.

Objective To investigate the protective effect of taurine on HK-2 cells exposed to oxalate (Ox) and calcium oxalate monohydrate crystal (COM) in vivo.Methods HK-2 cells,a proximal tubular epithelial cell line,were cultured.Five groups were divided in this study:control group (only HK-2 cells) ; Ox and COM group (HK-2 cells + Ox + COM) ; Taurine group (HK-2 cells + Ox + COM + Taurine) ; Apocynin group (HK-2 cells + Ox + COM + Apocynin) ; Catalase group (HK-2 cells + Ox + COM +Catalase).After 6 hrs,the cultures medias from each group were tested for LDH,H2O2,8-isoprostane,and MCP-1 protein.Cellular expression of MCP-1 mRNA and P47phox mRNA were determined by reverse transcriptase-polymerase chain reaction.After 24 hrs,cells livability was investigated by MTT.Results Compared with the control,cells livability was reduced when exposed to Ox and COM (P ＜ 0.05),Treatment with Taurine,Apocynin and Catalase significantly increased the cells livability (P ＜ 0.05).Compared with the control,the expression of LDH,H2O2,8-isoprostane,and cellular expression of MCP-1 mRNA and P47phox mRNA were increased following exposure to Ox and COM (P＜0.01,P＜0.01,P＜0.01,P＜0.01,P ＜0.05).Treatment with Taurine,Apocynin and Catalase significantly reduced the expression of LDH,H2O2,8-isoprostane,as well as the cellular expression of MCP-1 mRNA.Expression of P47phox mRNA in Taurine group was not reduced significantly (P ＞ 0.05).Conclusions This study showed that Taurine protected the HK-2 cells from oxidative injury exposed to Ox and COM by the pathway that may not be in relation to the inhibition of P47phox mRNA expression.

Objective To investigate the mechanism of melamine-induced renal damage in rats.Methods 48 male SD rats were randomly divided into 4 groups with 12 in each group and feed for 3 months.Group A were the control group,feed with standard granule feedstuff and drinking tap water.Group B were stone-induced group,feed with granule feedstuff containing 3％ Mel and drinking tap water.Group C were feed with granule feedstuff containing 3％ Mel and drinking water containing 2％ taurine.Group D were feed with standard granule feedstuff and drinking water containing 2％ taurine.Every week 24 h urine was collected to test PH,SCr,uric acid,protein,8-IP,H2O2 and Mel level.All rats were sacrificed at the end of 3 months.Blood creatinine detection,renal pathology analysis ( HE and Oil ep-red O dyeing,immunohistochemical) and mitochondria separation and detection were undertaken. ResultsMel was not detedted in urine of Group A and Group D.The urine concentration of Mel in Group B and Group C in 1 week,2 weeks,3 weeks,4 weeks were 3.16 ±0.45,4.39 ±0.213,5.40 ±0.28,5.50 ±3.26 and 3.52 ±0.49,4.32 ± 0.135,5.34 ± 0.40,5.46 ± 2.99 mg/ml,respectively.Compared with Group A,the Mel concentration in urine of Group B and C were drug exposure time dependent.In Group A,the urine protein,urine creatinine clearance,serum creatinine,and renal/weight ratio were 6.45 ± 1.45 mg/24 h,28.0 ± 7.4mmol/l,0.56 ±0.03 ml · min-1 · 100g-1,2.29 ±0.89 mg/g,while in Group B and C,the urinary protein urine,serum creatinine,creatinine clearance,kidney/weight ratio were 14.56 ± 7.69,56.8 ± 5.2,0.29 ±0.05,4.16 ±0.27 and 16.44 ±6.29,55.8 ±7.4,0.30 ±0.07,4.40 ±0.56,respectively.Compared with group A,in Group B and C,the urinary protein increased significantly,urine creatinine clearance reduced,serum creatinine reduced,and renal/weight ratio increased.Compared with Group B,the improvement of renal function in Group C was not significant,and the decrease of serum creatinine and urinary protein were not obvious (P ＞ 0.05).In Group B and C,the urine H2O2,8-IP and mitochondrial oxidatie detection reagent SOD,GSH-PX numerical were 28.5 ± 5.2 mmol/1,3.26 ± 1.6 pg/ml,21.1 ± 7.8 U/mg prot,19.0 ±2.5 energy unit and 26.7 ±4.8 mmol/l,2.99 ±8.5 pg/ml,20.3 ±6.9 U/mg prot,17.9 ±4.8 energy unit,respectively.The difference between Group B and C was not statistically significant (P ＞0.05).Pathological analysis showed Mel was mainly concentrated in crystal tubular lumen (Group B and C),kidney interstitial damage was apparent,and kidney inflammation and fibrosis progressive developed with the increase of the drug exposure time. Conclusions Mel can induce kidney damage and stone formation in rats,and stone was mainly in tubular location in inner medullary zone.It is not the oxidative stress way that Mel leads to kidney damage.

ObjectiveTo observe the interaction of the calcifying nanoparticles (CNP) with human renal tubular epithelial cells (HK-2) in vitro,to observe and investigate the mechanisms of HK-2 injury induced by CNP,and to explore the potential role of CNP in the formation of Calcium oxalate kidney stones.MethodsHuman renal tubular epithelial cells were cultured in vitro and CNP was then added to the culture medium,the cell-crystal reaction was detected by light microscopy and transmission electron microscopy (TEM).To investigate the oxidative stress,NADPH oxidase inhibitor apocynin was chosen as the intervener.The levels of LDH,MDA,HA in the mediums after 24 h were assessed. ResultsCNP could induce changes of the HK-2.Adhesion and phagocytosis of CNP by the HK-2 were observed under TEM.After 24 h,the levels of LDH,MDA,HA were significantly different among the 4 groups ( P ＜ 0.05 ). ConclusionsHK-2 has abilities of adhering and phagocyting with CNP.CNP can cause damage induced by oxidative stress of HK-2.

Objective To investigate the mechanisms of calculus crystal transport by macro-phage in kidney. Methods Hyperoxaluria rat model was established by administration of 1% ethyl-ene glycol and 1% ammonium chloride in drinking water. 24 h rat urine was collected, urinary oxalate were analyzed by ion chromatography. The expression and location of osteopontin and macrophage in kidney were observed by immunohistochemistry. Macrophage and calculus crystal at the basement membrane of renal tubular epithelial cells and interstitium were observed. Results The urinary ox-slate concentration were (0.22±0.13), (0.29±0.08), (0. 50±0.26), (0. 41±0. 22), (0.25±0. 12) ng/ml among these 5 groups. The osteoponitin expression was 0.16±0.04, 0.25±0.09, 0.37±0.10, 0.23±0.08, 0.19±0.02 respectively. The expression of osteopontin was positively correlated with urinary oxlate concentration(r=0.887, P<0.05). The macrophage at the basement membrane of renal tubular epithelial cells was 0.12±0.08, 0.19±0.06, 0.27±0.04, 0.16±0.03, 0.18±0.03 respectively. The macrophage distribution was positively correlated with the expression of osteopontin (r= 0.596, P<0.05). The macrophage moved from vessel to the basement membrane of loops of Henle, then disrupted and released the calculus crystal. Conclusions The macrophage might take part in the calculus crystal transport in kidney at the basement membrane of loops of Henle, which may be the source of Randall plaque. This process may be mediated by osteopontin.

BACKGROUND: Acellular amniotic membrane used widely in treating ocular surface disease as well as extensive burn wounds due to its low antigenicity and excellent histocompatibility. However, it is poorly understood whether it can be used in repairing urethral defects.OBJECTIVE: To evaluate role and the feasibility of human acellular amniotic membrane (HAAM) in the rabbit urethral reconstruction.DESIGN, TIME AND SETTING: The randomized control experiment of animals was performed at the Laboratory Center of Guangxi Medical University between April and June 2007.MATERIALS: Thirty-two New Zealand rabbits were supplied by animal center of Guangxi Medical University. The human HAAM was obtained from Department of Obstetrics, First Affiliated Hospital of Guangxi Medical University.METHODS: HAAM was prepared by detergent-enzymatic approach. Firstly, the fresh amniotic membrane was protected with cross linking of 1% formaldehyde- 0.2% glutaral, digested with 0.125% trypsogen- 0.05 mol/L EDTA, followed by washing with 0.5% Triton X-100. Totally 32 New Zealand male rabbits were assigned into 3 groups: experimental group (n=12), control group (n=12) and sham operation group (n=8). Rabbits were prepared for urethral defects models in the experimental and control groups, which were repaired with HAAM or anastomosised directly. There was no urethral operation in the sham operation group.MAIN OUTCOME MEASURES: The growth of epithelial cells and smooth muscle cells, as well as inflammatory cell infiltration was observed by histopathologic examination at days 10, 21 and 42 after operation. The urethral pressure changes and urinary bladder was examined by retrograde urethrography at day 42 after operation.RESULTS: ①The prepared HAAM was translucent, there was no residual cells or fragments. ②The pathological section examination showed that in the experimental group, some epithelial cells has grown without acute rejection at day 10 after operation, and several layers of urothelium covered HAAM at day 21 with reduced inflammatory cell infiltration. At day 42, a sprinkle of smooth muscle grew in HAAM with few inflammatory cells. Urodynamic studies indicated that there were no significant difference among 3 groups in the bladder volume, maximum urethral pressure and minimum urethral pressure (P > 0.05). The weight of bladder had obvious difference between the sham operation and control groups (P < 0.05).CONCLUSION: HAAM is an ideal biomaterial with well histocompatibility, biocompatibility and low antigenicity. HAAM is a good choice for urethral reconstruction.