Objective To determine the surfactant protein A (SP-A) subtype distribution and expression in human renal tissue and cultured human renal tubular epithelial cells(HK-2), and to explore the influence of Lipopolysaccharide (LPS) on the expression of SP-A subtypes mRNA and SP-A protein. Methods lmmunohistochemical staining was performed using SP-A polyclonal antibodies. RT-PCR was performed with mRNA from HK-2 cells and normal human kidney.Restriction fragment length polymorphism(RFLP) and sequencing were used to evaluate the subtypes of SP-A. The relative content of SP-A mRNA in human kidney and human lung was compared by real-time PCR. Western blotting analysis for SP-A was performed on protein from renal tissue and cultured HK-2 cells SP-A protein in human urine and culture supernatant of HK-2 cells was measured by enzyme-linked immunosorbent assay (ELISA) and Western blotting respectively. HK-2cells were treated with LPS at various concentrations (0,0.1,1,2,5,10 mg/L) for 8 h and at 5mg/L for various time points (0,2,4,8,16,24 h). Expression of SP-A mRNA and protein was analyzed by RT-PCR and Western blotting. Results SP-A was localized in renal tubular epithelial cells of both proximal and distal convoluted tubules. SP-A1, SP-A2 mRNA and protein could be detected in normal HK-2 cells and human kidney. The significant secretion of SP-A [urine: (106.614172.772) nmol/L, n=30; culture supernatant: (85.533±58.622) nmol/L, n=10] was shown. The levels of SP-A1, SP-A2 mRNA and Sp-A protein in HK-2 cells were significantly decreased after treatment with LPS. Conclusions Human renal tubular epithelial cells can express both SP-A1 and SP-A2 genes which may play an important role in inflammation modulation of kidney.

Objective To investigate the effect of surfactant protein D(SP-D)overexpression on lipopolysaccharide(LPS)-induced monocyte chemoattractant protein-1(MCP-1)expression in human renal proximal tubular epithelial cells(HK-2)and its mechanism. Methods HK-2 cells were treated with LPS at various concentrations (0, 0.1, 1, 2, 5, 10 mg/L)for 8 h and at 5 mg/L for various time points(0, 2, 4, 8, 16, 24 h). Expression of SP-D was detected by Western blotting and real-time PCR. Expression of MCP-1 was determined by ELISA and real-time PCR. Human SP-D cDNA eukaryotic expression vector pEE14-hSP-D was transfected to HK-2 cells. The changes in transfected cells of SP-D protein were observed by Western blotting. Expression of MCP-1 was detected by ELJSA and real-time PCR. Results SP-D was expressed in HK-2 cells. The levels of SP-D protein and mRNA in HK-2 cells were significantly decreased after treatment with LPS(P<0.05). Expression of MCP-1 protein and mRNA was increased remarkably after treatment with LPS(P<0.05). HK-2 cells transfected with pEE14-hSP-D showed up-regulated expression of SP-D. The overexpression of SP-D inhibited the LPS-inducedexpression of MCP-1(P<0.01). Conclusions SP-D inhibits LPS-induced expression of MCP-1 in HK-2 cells. SP-D may play an important role in the modulation of renal inflammation.

Objective To investigate the effect of angiotensin 1-7(Ang 1-7) on renal proximal tubular epithelial cell(HK-2) transdifferentiation induced by high glucose.Methods All the raised HK-2 cells were divided into 5 groups: normal control group,high glucose group,high glucose with Ang1-7 group,high glucose with Ang1-7 and A779 group,high glucose with pioglitazone group.Expression of peroxisome proliferator activated receptor-γ(PPAR-γ) and α-smooth muscle actin(α-SMA) was detected by Western blotting,real-time PCR and immunofluorescence.Results The levels of PPAR-γ protein and mRNA in HK-2 cells were significantly increased after treatment with high glucose and Ang 1-7.Expression of α-SMA protein and mRNA was inhibited remarkably after treatment with high glucose and Ang 1-7.These effects of Ang 1-7 on HK-2 cells could be reversed by Mas receptor antagonist A779.Conclusion Ang 1-7 inhibits high glucose-induced expression of o-SMA in HK-2 cells,which is in part through the Mas.

Objective To investigate the role of surfactant protein (SP)-A and SP-D in urinary tract infection mouse model,and evaluate the effects of SP-A and SP-D absence on urinary tract infection.Methods SP-A and SP-D double knockout (SP-A/D KO) mice were made.SP-A/D KO and wild-type (WT) C57BL/6 female mice were used for this study.The expression of SP-A and SP-D in kidney was detected by immunohistochemistry (IHC).The levels of p-p38 and p38 protein in kidneys were measured by Western blotting.Uropathogenic Escherichia coli or buffer was delivered into the bladder of female mice.At 24 and 48 h after inoculation,CFU of Escherichia coli in the kidney and urine of the treated and control mice were measured.Histological,cellular and molecular analysis were performed by several methods of H/E staining,IHC and Western blotting.The effects of SP-A and SP-D on bacterial growth were studied in vitro.Results SP-A and SP-D in kidney were located in the proximal tubules and collecting tubules.Compared with WT mice,infected SP-A/D KO mice with UPEC had higher CFU in kidneys and urine at 24 h and 48 h,increased inflammatory cells infiltration in kidneys (P＜0.05).Compared with WT mice,SP-A/D KO mice had higher p38 MAPK phosphorylation levels in kidneys (P ＜ 0.05).Growth of Escherichia coli was greatly inhibited by both SP-A and SP-D (P＜0.05).Conclusions Both SP-A and SP-D are expressed in kidney.SP-A and SP-D can attenuate UTI induced by UPEC which may be through inhibiting bacterial growth and modulating renal inflammation.

Objective To evaluate the effect of aldosterone (Ald) on glomerular mesangial cells apoptosis and to explore the possible mechanisms.Methods Twenty-four Sprngue-Dawley rats were subcutaneously embedded with osmotic mini-pumps and randomly divided into 3 groups.Aldosterone (1.5 μg/h) was administrated subcutaneouly by osmotic mini-pumps in Ald group,eplerenone (Epl,100 mg·kg-1·d-1) and Ald (1.5 μg/h) was given to Epl group.And normal saline was used in control group (Con group).Systolic blood pressure and urinary albumin excretion rate (UAER) were detected on day 0,7,14,21,28.Blood and kidney samples were harvested on day 28.Plasma creatinine,potassium and aldosterone were measured.Renal paraffin sections were stained by PAS and the morphological changes were evaluated by light microscopy.Apoptosis index of mesangial cells were detected by TUNEL assay.The glomerular mesangial cells (MCs) were cultured in a DMEM-F12 media.MCs apoptosis was evaluated by staining cells with Annexin V and propidium iodide (PI) using flow cytometer.Expression of Bcl-2 and Bax mRNA was examined by RT-PCR.The protein level of Bad or phospho-Bad was measured by Western blotting.Results Ald-infused rats developed hyperaldosteronemia and hypokalemia.Rats in Ald group exhibited significant hypertension and marked albuminuria.Ald group rats showed increased number of TUNEL-positive mesangial cells when compared with control rats (P＜0.05).Aldosterone induced mesangial cells apoptosis in a time-dependent manner.Expression of Bcl-2 mRNA was decreased but Bax mRNA was increased in aldosterone treated MCs compared to that in Con group (P＜0.05).Aldosterone promoted dephosphorylation of cytosolic phospho-Bad compared with vehicle treated cells (P＜ 0.05).However,eplerenone attenuated these effects of aldosterone.Conclusion Aldosterone directly promotes mesangial cells apoptosis,and eplerenone can attenuate this effect of aldosterone.Dephosphorylation of cytosolic phospho-Bad may be the key role in the progression of mesangial cells apoptosis induced by aldosterone.

Objective To investigate the application of MSCT three-dimensional digital navigated biopsy in subcarinal lesions.Methods 82 patients were enrolled.Study subjects were randomly divided into control group and research group.Three-dimensional positioning and three-dimensional navigation needle biopsy were used in research groups, while CT cross-sectional image positioning with conventional puncture needle was used in control group.Puncture accuracy, one-time success rate of puncture, complications, diagnosis accuracy and operation time were compared between the two groups.Results Puncture success rate, definite diagnosis rate were 87.80%(36/41) and 97.56%(40/41) for the research group,and 60.97%(24/41) and 80.49% (33/41) for the control group, respectively,which on the research group were higher than that on the control group(χ2=8.945, 6.116;P<0.05).Complication rate and operating time were 14.63% (6/41) and (11.64±2.76) min for the research group, and 41.45% (17/41) and (22.22±6.31) min for the control group, respectively, which were lower on the research group than that on the control group (χ2=7.31,t=-11.70,P<0.05).Conclusion MSCT three-dimensional digital navigated biopsy technique could promote the efficiency of subcarinal space puncture biopsy significantly,which is a novel, convenient, precise and safe method.

OBJECTIVE:To provide reference for rational drug use in the clinic. METHODS:Irrational medication orders eval-uated by the Affiliated Cancer Hospital of Zhengzhou University during Oct. 2014 to Sep. 2015 were arrangemented,summarized and analyzed. RESULTS:A total of 515 inpatient medical records were reviewed and analyzed,among which there were 165 unrea-sonable medical records and 185 irrational medication orders. Irrational medical records of general surgery department were the most(38 items,accounting for 23.03%). Irrational drug use mainly included irrational usage and dosage(80.00%),drug use with-out indications or not suit indications (7.57%),inappropriate solvent selection (4.86%). Including 66.22% of single overdose, 18.92% of longer medication duration. CONCLUSIONS:There are many irrational medical orders which should be standardized in our hospital,especially overdose and longer medication duration,which increase financial burden of patient. Pharmacists should strengthen communication with clinicians,and hold rational drug use trainings regularly base on the types of the irrationality. These can help to improve rational drug use and guarantee the safety of drug use.

ObjectiveTo evaluate the effects of Ang Ⅱ on apoptosis of podocytes and explore the signaling pathwayof nephrin in preventingAng Ⅱ-inducedpodocyte apoptosis.MethodsDifferentiated mouse podocytes were exposed to Ang Ⅱ at different concentrations for 18 h or at 10-8 mol/L for variable incubation times.Undifferentiated mouse pedocytes were transfected using lipofectamine 2000 with the pcDNA3.1-mNPHS1 plasmid and stably transfected cell lines were generated with G418 selection.In separated experiments,untransfected mouse podocytes (MPC) and stably transfected podocytes with pcDNA3.1-neo and PcDNA3.1-mNPHS1 were exposed toAng Ⅱ(10-8 mol/L) or LY294002(a selective Akt inhibitor,50 μmol/L) for indicated times.Apoptosis was evaluated by flow cytometry.The expression of nephrin was assessed by quantitative real-time PCR,immunofluorescence and Western blotting.The phosphorylation level of Akt was determined by Westem blotting.Results(1) AngⅡ promoted podocyte apoptosis in a dose-and time-dependentmanner. PretreatmentwithlosartansignificantlypreventedAngⅡ -induced apoptosis. (2) Nephfin mRNA and protein were obviously decreased in podocytes exposed to 10-8 mol/L Ang Ⅱ for at least 12 h than those in vehicle-treated cells (P＜0.05).(3) Ang Ⅱ exposure for more than 15 min inhibited the phosphorylation of AKT in MPC,which was dramatically reversed by pcDNA3.1-mNPHS1 transfection,but not by pcDNA3.1-neo transfection. (4) Podocyte apoptosis was promoted byLY294002. Conversely,Ang Ⅱ-induced podocyteapoptosis was significantly alleviated by pcDNA3.1-mNPHS1 transfection.ConclusionAng Ⅱinduces mouse podocyte apoptosis which is suppressed by overexpression of nephrin through PI3K-Akt signaling pathway.

ObjectiveTo investigate whether aldosterone infusion induces glomerular or podocyte injury in rats and to evaluate the effects of eplerenoen (EPL), andodipine (CCB) and telmisartan (ARB) on aldosterone- induced injury. MethodsThirty male Sprague-Dawley rats were divided into 5 groups: control, subcutaneous infusion of aldosterone (1.5 μg/h, ALD group) and aldosterone infusion plus eplerenone (100 mg·kg-1·d-1, EPL group), amlodipine(10 nag·kg-1·d-1 CCB group), telmisartan (3 mg·kg-1·d-1, ARB group), respectively. Systolic blood pressure(SBP) and urinary albumin excretion ratio(UAER) were measured at day 0, 7, 14, 21, 28. Blood samples were harvested to detect plasma angiotensin Ⅱ, plasma aldosterone, serum sodium, serum potassium and serum creatinine at day 28. Glomerular damge was quantified by morphological glomerular injury score (GIS). Immunohistochemistry and RT-PCR were performed to evaluate podocyte lesion, and apoptosis ratio of pedocyte (ARP) in a glomerular cross section was analyzed by TUNEL. ResultsALD infusion progressively increased SBP and UAER compared with CTL (P＜0.01). SBP was significantly reduced in EPL, CCB or ARB-treated animals, meanwhile, UAER was decreased in EPL and ARB group, but not in CCB group. The ALD-infused rats exibited hypernatremia and hypopotassaemia, which were blocked by EPL adminstration but not by CCB or ARB treatment. ARB group had a significant increase in plasma angiotensin Ⅱ compared with ALD, CCB and EPL groups(P＜0.01). The ALD-infused animals developed hyperaldosteronemia compared with CTL, but with no difference of plasma aldosterone among ALD, EPL, CCB and ARB-treated rats. Treatment with EPL prevented an increase of GIS and ARP compared with CCB and ARB (P＜0.05, P＜0.01). Protein and mRNA expression of nephfin was up-regulated in ALD group (P＜ 0.01), but was significantly prevented by EPL treatment(P＜0.01), whereas CCB and ARB therapy had no such effect. Conclusion ALD infusion significantly induces glomerular and pedocyte injury which is blocked by EPL but not by CCB or ARB independently on systemic hemodynamics.

Objective To investigate the correlation between asymmetric dimethylarginine (AD-MA) and endothelial dysfunction in patients with uremia.Methods Uremic patients who did not receive hemodialysis were defined as A group (n =40) ; uremic patients who had received hemodialysis were divided into B group (n =45) ;healthy people were defined as C group (n =20) ;and chronic kidney disease (stage 2 ～ 4) patients were defined as D group (n =20).The diameter of intima-media thickness,and endothelium-dependent or independent dilation (EDD or EID) of radial artery in right forearm were detected with diasonography.The levels of ADMA were determined by high-performance liquid chromatography.Results Compared to C group,the levels of ADMA in A,B and D groups were significantly increased [C:(0.78 ±0.19) μmol/L,A:(1.51 ±0.16) μ mol/L,B:(1.13 ±0.14) μmol/L,D:(0.92 ±0.11) μmol/L; P ＜0.05].Compared to A group,the levels of ADMA were significantly decreased in B group (P ＜0.05).EDD and EID were decreased significantly in A,B and D groups compared to C group [EDD:C:(13.52±1.73)％ vs A:(7.32 ±0.54)％,B:(9.02 ±0.86)％,D:(10.13 ±1.25)％,P ＜0.05;EID:C:(14.45±1.85)％ vsA:(10.37 ±1.51)％,B:(9.54±1.39)％,D:(11.17±1.56)％,P ＜0.05].EDD in B group was significantly lower than A group (P ＜0.05).In group A,a negative correlation was found between EDD and the level of ADMA (r =-0.81,P =0.020).Conclusions ADMA level was significantly increased in uremic patients.A close correlation existed between ADMA and endothelial dysfunction of radial artery.