Role and mechanism of C3a-C3a receptor in autosomal dominant polycystic kidney disease

Objective To explore the role and mechanism of C3a-C3a receptor (C3aR) in the progression of autosomal dominant polycystic kidney disease (ADPKD). Methods Renal tissues of ADPKD patients and PKD1 knockout mice were collected. Then the expression of C3a-C3aR, Ki67 and F4/80 in renal tissues was observed. Macrophages were stimulated with lipopolysaccharide (LPS) and interleukin 4 respectively. The expression of C3aR, TNF-α, typing markers and related signal pathway proteins was detected in each group. PKD1 knockout mice were treated with C3aR inhibitor SB290157 (1 mg/kg). Renal pathology, cyst-related indicators and renal function were observed. Results The expression of C3a and C3aR in ADPKD was up-regulated (both P＜0.05); C3aR and F4/80 were co-located in the kidney of polycystic kidney disease (PKD) mice, indicating that C3aR was mainly expressed on membrane of macrophages. In vitro, the expression of C3aR was up-regulated in M1 macrophages (P＜0.05). After the stimulation of C3a, the expression of iNOS, TNF-α and IL-6 mRNA in M1 macrophages were up-regulated (all P＜0.05), as well as the secretion of TNF-α, indicating that C3a not only affected the expression of inflammatory factors of M1 macrophages, but also affected the inflammatory microenvironment. In addition, C3a significantly activated Akt in M1 macrophages (P＜0.05). Compared with the control group, the treatment group showed a decrease in C3a-C3aR as well as serum BUN, Scr, cyst index, and two kidneys weight/body weight (2KW/BW) (all P＜0.05), and ADPKD related pathway protein expression such as p-ERK and p-P65 was significantly down-regulated (all P＜0.05). Conclusions The increased C3a in polycystic kidney tissue causes infiltration and activation of macrophages through C3aR, and then promotes ADPKD progression. The mechanism may be mediated by Akt activation and increased TNF-α production. C3aR antagonist is a potential research direction in the treatment of ADPKD.     

Expression of apoptosis stimulating protein two of p53 in acute kidney injury induced by carbon tetrachloride in mice

Objective To investigate the expression of apoptosis stimulating protein two of p53 (ASPP2) in acute kidney injury (AKI) induced by carbon tetrachloride (CCl4) in mice. Methods Thirty-two male Balb/c mice were randomly divided into olive oil control group (control group, 8 mice) and CCl4 experimental group (experimental group, 24 mice). A mouse model of AKI was induced by a single high-dose abdominal injection of CCl4. The mice in the experimental group were sacrificed at 12 h, 24 h and 48 h after CCl4 injection. The mice in the control group were sacrificed 24 h after treatment. The serum and kidney tissue samples were collected. Serum biochemical indicators [serum creatinine (Scr) and urea nitrogen (BUN)] were measured by automatic biochemical analyzer. The pathological damage of kidney tissue was observed by hematoxylin-eosin (HE) staining and Periodate-Schiff (PAS) staining. ASPP2 positioning and expression level were observed by immunohistochemistry. The apoptosis of mouse kidney tissue was detected by in situ apoptosis. The expression of ASPP2 protein and ASPP2 mRNA in renal tissue were detected by Western blotting and quantitative real-time PCR. Results Compared with the control group, the levels of Scr and BUN were significantly increased in the experimental group (P＜0.01). Histopathology showed partial renal tubular brush margin detachment, renal tubular epithelial cell necrosis and nuclear disintegration in the experimental group. TUNEL staining showed that the apoptosis rate of renal tissue cells increased significantly in the experimental group (P＜0.01). Compared with the control group, the expression of ASPP2 in the experimental group increased at the early period and then decreased with the prolongation of injury time. The mRNA expression was consistent with the protein expression, and all reached the peak after 24 hours injury (P＜0.01). Immunohistochemistry showed that ASPP2 was mainly localized in the cytoplasm of renal tubular epithelial cells. Conclusions A single high-dose injection of CCl4 in the abdominal cavity can induce AKI in mice. The expression of ASPP2 is consistent with the degree of renal tissue damage. As the damage of renal tissue is aggravated, the expression of ASPP2 is gradually increased, which indicates that ASPP2 may be a damage factor.     

Effect of a C5a receptor antagonist on macrophage function in an intestinal transplant rat model

BackgroundC5a promotes alloreactivity via the C5a receptor 1 (C5aR1) on immune cells, but this has not been confirmed in the case of small intestine transplantation immunity. In the present study, we examined the effect of C5aR1 antagonist (PMX53) on macrophage function in small intestinal transplantation.MethodsThe model was created by heterotopic intestinal transplantation using donor Dark Agouti and recipient Lewis rats. PMX53 was administered starting on the day of operation until postoperative day 7. The graft survivals were compared, and HE staining of grafts, lymphocyte mixed reaction test (MLR, mixed culture of T cells from lymph nodes and spleen cells from donors), and changes in macrophage and T cell accumulation in grafts on day 6 after transplantation were evaluated. In addition, the effect of PMX53 on macrophage differentiation and activation was assessed using macrophages derived from bone marrow (BMDM).ResultsGraft survival was significantly prolonged in the therapeutic group compared to the untreated group. Histological evaluation showed that PMX53 inhibited the shortening of the graft villus, and the stimulation index of MLR was significantly lower in the therapeutic group compared to the untreated group. In the therapeutic group, the accumulation of macrophages in intestinal graft and monocyte in blood were reduced, compared with the untreated group. PMX53 decreased the differentiation in BMDM and the mRNA expression of IL-1β and TNF-α in activated BMDM.ConclusionInhibition of C5a/C5aR1 signaling appears to regulate macrophage differentiation and suppress rejection in small intestine transplantation immunity.     

C3a and c5a promote renal ischemia-reperfusion injury

Renal ischemia reperfusion injury triggers complement activation, but whether and how the small proinflammatory fragments C3a and C5a contribute to the pathogenesis of this injury remains to be elucidated. Using C3aR-, C5aR-, or C3aR/C5aR-deficient mice and models of renal ischemia-reperfusion injury, we found that deficiency of either or both of these receptors protected mice from injury, but the C3aR/C5aR- and C5aR-deficient mice were most protected. Protection from injury was associated with less cellular infiltration and lower mRNA levels of kidney injury molecule-1, proinflammatory mediators, and adhesion molecules in postischemic kidneys. Furthermore, chimera studies showed that the absence of C3aR and C5aR on renal tubular epithelial cells or circulating leukocytes attenuated renal ischemia-reperfusion injury. In vitro, C3a and C5a stimulation induced inflammatory mediators from both renal tubular epithelial cells and macrophages after hypoxia/reoxygenation. In conclusion, although both C3a and C5a contribute to renal ischemia-reperfusion injury, the pathogenic role of C5a in this injury predominates. These data also suggest that expression of C3aR and C5aR on both renal and circulating leukocytes contributes to the pathogenesis of renal ischemia-reperfusion injury.     

肾小管上皮细胞程序性坏死对慢性肾脏病患者肾损伤的影响

目的:分析不同分期慢性肾脏病（chronic kidney disease,CKD）患者肾组织中发生程序性坏死的肾小管上皮细胞数量及其与临床病理指标的相关性,探讨肾小管上皮细胞程序性坏死在CKD患者肾小管上皮细胞过度死亡、慢性肾损伤进展中的作用。方法:收集2017年6月至2019年6月于海南医学院第一附属医院行肾活检且...     

Effect of thymosin β4 on transforming growth factor-β/connective tissue growth factor of renal tubular interstitial fibrosis rats

Objective To investigate the influence of thymosin beta 4 (Tβ4) with two different dosages on the expression of transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) in rats with renal tubular interstitial fibrosis, and to further estimate the changes of renal tubular interstitial lesions. Methods Rat models of renal tubular interstitial fibrosis were established by unilateral ureteral occlusion (UUO). The male SD rats were randomly divided into 4 groups and 15 rats in each group: sham group, model group, treatment group with 1 mg/L Tβ4 and treatment group with 5 mg/L Tβ4. Rats in sham group and model group were poured into the same amount of saline. The renal function and renal pathological changes were observed after the second week. The mRNA and protein expression of TGF-β and CTGF in renal tissues was tested by in-situ hybridization and Western blotting. Results Compared with that in sham group, the expression of TGF-β mRNA and its protein, CTGF mRNA and its protein was significantly higher in model group (all P＜0.01). Compared with rats of model group, Tβ4 treatment rats had lower mRNA and protein expression of TGF-β and CTGF (all P＜0.01), and the expression in treatment group with 5 mg/L Tβ4 was lower than that in treatment group with 1 mg/L Tβ4 (P＜0.05). And the expression of TGF-β mRNA was positively correlated with CTGF mRNA expression (r=0.697, P＜0.01). The 24 h total urinary protein and the area of renal tubular interstitial lesion in model group were significantly more than those in sham group, and also more than those in Tβ4 treatment group (all P＜0.05). Tβ4 treatment attenuated kidney damage, and the effects in treatment group with 5 mg/L Tβ4 were better than those in treatment group with 1 mg/L Tβ4. No difference in serum creatinine and blood urea nitrogen was observed among 4 groups (all P＞0.05). Conclusions Tβ4 treatment can inhibit the renal TGF-β and CTGF expression of rats with tubular interstitial fibrosis in a dose-dependent manner, and play a protective role in kidney.     

补体C5抑制剂对急性胰腺炎大鼠肾细胞因子的调控作用

目的 观察补体C5抑制剂对急性胰腺炎大鼠肾细胞因子的调控作用.方法 Wistar大鼠42只,随机分为3组:正常对照组(HC组,n=6);AP造模组(AP组,n=18);AP造模+C5抑制剂组(AP-C5I组,n=18).分别在6、12、18 h三个时间点处死AP组、AP-C5I组各6只.分别测定其血C5a水平;采用实时荧光定量逆转录.聚合酶链反应(RT-PCR)方法测定各组大鼠肾组织中C5、IL-1β、TNF-α和IL-10 mRNA表达情况.结果 AP组血清C5a水平显著升高(P＜0.01、vs HC);AP-C5I组其水平显著降低(P＜0.01 vs AP).HC组肾内C5 mRNA表达较弱;AP组该值较HC组有显著增高,差异有统计学意义(P＜0.01 vs HC);AP-C5I组肾组织C5 mRNA表达较AP组无显著改变(P＞0.05 vs AP).AP组.肾组织IL-β、TNF-αmRNA在6 h明显增多并持续增至18 h(P＜0.01 vs HC),AP-C5I组两值较同时相AP组明显降低(P＜0.01 vs AP);AP组和AP-C5I组肾组织IL-10 mRNA水平于建模后先升后降,AP-C5I组于6 h和12 h较AP组差异有统计学意义(P＜0.05 vs AP).结论 补体C5抑制剂能够阻断补体系统的激活,并部分恢复肾组织促抗炎细胞因子平衡.     

Effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells

Objective To evaluate the effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells and to explore the possible mechanisms of KIM-1 involved in renal interstitial fibrosis of DN. Methods The rat renal tubular epithelial cells (NRK52E) were cultured in vitro and divided into five groups: Normal control group (D-glucose 5.6 mmol/L), Hypertonic group (D-glucose 5.6 mmol/L＋D-mannitol 24.4 mmol/L), High glucose group (D-glucose 30 mmol/L), Control siRNA group,KIM-1 siRNA group. ELISA assay was used to assess the levels of MCP-1 and FN in the cells supernatant; Western blotting was used to detect the protein expression of KIM-1; RT-PCR was used to detect mRNA expression of KIM-1, MCP-1 and FN. Results Compared with the control group, the protein and mRNA expression of KIM-1 in the high glucose group were increased at 12 h (P＜0.05), and reached the peak at 48 h (P＜0.05); the protein and mRNA expression of MCP-1 and FN in high glucose group were increased at 24 h significantly (P＜0.05), and peaked at 48 h (P＜0.05). Compared with the high glucose group, the protein and mRNA expressions of MCP-1 and FN in KIM-1 siRNA group were decreased (P＜0.05). Conclusions Down-regulating the expression of KIM-1 can significantly inhibit the expression of MCP-1 and FN, which suggests that KIM-1 may be involved in renal interstitial fibrosis of DN by regulating expression of MCP-1 and FN.     

Complement Mediated Renal Inflammation Induced by Donor Brain Death: Role of Renal C5a‐C5aR Interaction

Kidneys retrieved from brain‐dead donors have impaired allograft function after transplantation compared to kidneys from living donors. Donor brain death (BD) triggers inflammatory responses, including both systemic and local complement activation. The mechanism by which systemic activated complement contributes to allograft injury remains to be elucidated. The aim of this study was to investigate systemic C5a release after BD in human donors and direct effects of C5a on human renal tissue. C5a levels were measured in plasma from living and brain‐dead donors. Renal C5aR gene and protein expression in living and brain‐dead donors was investigated in renal pretransplantation biopsies. The direct effect of C5a on human renal tissue was investigated by stimulating human kidney slices with C5a using a newly developed precision‐cut method. Elevated C5a levels were found in plasma from brain‐dead donors in concert with induced C5aR expression in donor kidney biopsies. Exposure of precision‐cut human kidney slices to C5a induced gene expression of pro‐inflammatory cytokines IL‐1 beta, IL‐6 and IL‐8. In conclusion, these findings suggest that systemic generation of C5a mediates renal inflammation in brain‐dead donor grafts via tubular C5a‐C5aR interaction. This study also introduces a novel in vitro technique to analyze renal cells in their biological environment.     

外源性硫化氢在大鼠肾脏缺血再灌注损伤中的保护作用

目的 观察不同剂量外源性硫化氢(H2S)供体硫氢化钠对大鼠肾脏缺血再灌注损伤( IRI)的保护作用.方法 健康雄性Wistar大鼠28只随机分为4组,即假手术组( Sham)、肾缺血再灌注(IR)组、硫氢化钠(NaHS)高剂量组、硫氢化钠低剂量组.大鼠右肾切除后,以NaHS作为硫化氢的供体,NaHS高、低剂量组分别经左肾动脉插管,按照1.5 μmol/min、300 nmol/min的剂量连续15 min给药,假手术组及IR组给予同体积生理盐水.停药5 min 后,NaHS组和IR组用无损伤微动脉夹夹闭左侧肾蒂45 min后解除阻断,建立大鼠急性IRI模型,假手术组不夹闭左肾动脉,其他操作同模型组.于肾脏恢复血流24h时留取血和肾组织标本,检测血清尿素氮(BUN)、血肌酐(Scr);半定量分析肾脏病理损伤;检测肾组织H2S生成率;采用实时定量PCR法检测胱硫醚-β-合成酶(CBS)、胱硫醚-γ-裂解酶(CSE )mRNA表达.结果 与假手术组相比,IR组H2S生成率显著降低(P＜0.01);CBS、CSE mRNA表达显著下降(P＜0.01 );Scr、BUN显著升高(P＜0.01);肾脏病理表现为急性肾小管坏死,且最严重.与IR组相比,NaHS预处理组H2S生成率升高(P＜0.05);CBS、CSE mRNA表达升高(P＜0.01 );Scr、BUN降低(P＜0.01);病理损伤明显减轻.NaHS两个剂量组之间差异无统计学意义.结论 外源性H2S对大鼠IRI具有保护作用.     

Complement 5a receptor inhibition improves renal allograft survival

Complement activation plays a key role in mediating apoptosis, inflammation, and transplant rejection. In this study, the role of the complement 5a receptor (C5aR) was examined in human renal allografts and in an allogenic mouse model of renal transplant rejection. In human kidney transplants with acute rejection, C5aR expression was increased in renal tissue and in cells infiltrating the tubulointerstitium. Similar findings were observed in mice. When recipient mice were treated once daily with a C5aR antagonist before transplantation, long-term renal allograft survival was markedly improved compared with vehicle-treatment (75 versus 0%), and apoptosis was reduced. Furthermore, treatment with a C5aR antagonist significantly attenuated monocyte/macrophage infiltration, perhaps a result of reduced levels of monocyte chemoattractant protein 1 and the intercellular adhesion molecule 1. In vitro, C5aR antagonism inhibited intercellular adhesion molecule 1 upregulation in primary mouse aortic endothelial cells and reduced adhesion of peripheral blood mononuclear cells. Furthermore, C5aR blockade markedly reduced alloreactive T cell priming. These results demonstrate that C5aR plays an important role in mediating acute kidney allograft rejection, suggesting that pharmaceutical targeting of C5aR may have potential in transplantation medicine.     

Expression of secreted frizzled related protein 4 and effect of induced-apoptosis in autosomal dominant polycystic kidney disease

Objective To evaluate the expression of secreted frizzled related protein 4 (sFRP4) in autosomal dominant polycystic kidney disease(ADPKD) and the effect of sFRP4 induced apoptosis in ADPKD. Methods (1)Serological method: serum samples of 12 healthy people and 20 ADPKD patients were collected and the levels of sFRP4 in serum were detected by ELSIA assay. (2)Tissue experiments: normal renal tissue was collected from radical nephrectomy for renal carcinoma; polycystic renal tissues were taken from ADPKD patients. The expression of sFRP4 in renal tissues was observed by immunohistochemistry; Real-time PCR was used to explore the mRNA level of sFRP4 and caspase-3; TUNEL method was applied to observe the apoptosis cells existing in ADPKD. (3)studies in vitro: HEK-293T cells were transfected with PcDNA6 and Flag. sFRP4.PcDNA6 respectively, after which Western blotting was performed to detect the expression of caspase-3 protein and flow cytometry was performed to estimate cell apoptosis rate. Results (1)ELISA results showed serum concentrations of sFRP4 in ADPKD were markedly higher than normal control (P＜0.05). (2)Compared with normal renal tissues, the sFRP4 expression was dramatically increased in ADPKD and mainly distributed in the cytoplasm of renal tubular epithelial cells. (3)Real-time PCR showed the expression of sFRP4 and Caspase-3 mRNA in ADPKD were up-regulated comparing with those in normal control (P＜0.05). (4)TUNEL assays revealed that elevated apoptosis appeared in tubular epithelial cells of ADPKD. (5)The level of caspase-3 protein and apoptosis rate were significantly increased after over-expressed sFRP4 in HEK-293T cells (all P＜0.05). Conclusions The expression of sFRP4 is strikingly up-regulated in ADPKD. In addition, abnormal apoptosis of tubular epithelial cells exists in ADPKD and over-expressed sFRP4 can induce apoptosis of HEK-293T cells. This phenomenon may be attributed to the elevated sFRP4.     

氧化应激介导糖尿病大鼠肾小管上皮细胞转分化及普罗布考的干预作用

目的 研究氧化应激在糖尿病肾病（DN）大鼠肾小管上皮细胞转分化中的作用,探讨抗氧化剂普罗布考对大鼠DN的肾脏保护作用。 方法 30只雄性SD大鼠随机分为正常对照组、DN组和普罗布考干预组（1％普罗布考饮食）,每组10只。分别于第3周、第8周及第12周检测24 h尿蛋白（UTP）;12周末检测各组大鼠血糖、血脂（胆固醇、三酰甘油）、Scr、肌酐清除率（Ccr）、肾脏组织匀浆液丙二醛（MDA）含量及谷胱甘肽过氧化物酶（GSH-Px）活性。肾组织病理切片行 HE和Masson染色;采用免疫组化和Western印迹检测肾组织核转录因子Sp1、α平滑肌肌动蛋白（α-SMA）及E钙黏蛋白（E-cadherin）表达。 结果 与正常对照组比较,DN组血糖、Scr、肾组织匀浆MDA和24 h UTP水平显著增高（均P < 0.01）,Ccr显著降低（P < 0.01）;肾组织肾小管损伤分数、α-SMA和 Sp1蛋白表达水平明显增高（均P < 0.01）;肾组织E-cadherin蛋白表达明显下调。肾组织MDA含量分别与α-SMA及Sp1蛋白表达呈正相关（r ＝ 0.896,P < 0.01;r ＝ 0.862,P < 0.01）,与E-cadherin蛋白表达呈负相关（r = -0.673, P < 0.01）。普罗布考干预组Scr、24 h UTP、肾组织MDA、肾小管损伤分数及肾组织α-SMA、 Sp1蛋白表达水平较DN组均明显降低（均P < 0.01）;Ccr和肾组织E-cadherin蛋白表达水平较DN组均明显增加（均P < 0.01）。 结论 氧化应激在DN大鼠肾小管上皮细胞转分化中起重要作用。普罗布考可能通过抗氧化、下调肾组织Sp1蛋白表达及抑制肾小管上皮细胞转分化延缓DN大鼠肾脏病变进展。     

Expression of tumor necrosis factor-α induced protein 8 like-1 in cisplatin-induced acute kidney injury models

Objective To investigate the expression and role of the tumor necrosis factor-α (TNF-α) induced protein 8 like-1 (TIPE1) in acute kidney injury (AKI) induced by cisplatin in animal model and cells. Methods Twelve male C57BL/6 mice aged 6-8 weeks were randomly divided into the control group and the model group. Mice in the model group received a single intraperitoneal injection of 20 mg/kg of cisplatin (20 mg/kg saline in the control group). All mice were euthanized after 5 days. Meanwhile, serum and kidney samples were collected. The levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were detected by biochemical kits. Renal histopathological changes in mice were observed by HE staining. The expression of TIPE1 in kidney was examined using immunohistochemistry. qRT-PCR was used for testing the relative expression of TIPE1 mRNA in mice kidney. Western blotting was used for testing TIPE1 and NGAL protein relative expression in mice kidney. Human kidney proximal tubular cells (HK-2) were stimulated with 20 μmol/L cisplatin for 0, 6, 12 and 24 h to establish cisplatin-induced AKI cell model. The expressions of TIPE1 mRNA and protein were detected by qRT-PCR and Western blotting in HK-2 cells. The expression of TIPE1 gene in HK-2 cells was silenced by lentivirus containing TIPE1 siRNA sequence. Then, TIPE1 stable knockout HK-2 cell strains were treated with 20 μmol/L of cisplatin for 24 hours. The protein expression of tubular damage marker neutrophil gelatinase-associated lipocalin (NGAL), microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 in HK-2 cells were detected by Western blotting. Results Compared with the control group, the expressions of TIPE1 mRNA and protein were up-regulated and NGAL protein expression was increased significantly in renal tissue of the model group (all P＜0.05). The expressions of TIPE1 mRNA and protein were remarkably increased with the prolongation of cisplatin treatment in HK-2 cells (both P＜0.05). Compared with the scramble siRNA group, the protein expressions of NGAL, LC3-Ⅱ and Beclin1 were increased significantly in the TIPE1 siRNA group after lentivirus interfered with the expression of TIPE1 gene in HK-2 cells (all P＜0.05). Conclusions The mRNA and protein expressions of TIPE1 are increased in acute kidney injury models. Gene silencing of TIPE1 can promote the expressions of early renal tubular damage marker and autophagy-related proteins, which indicates the excessive autophagy aggravates renal tubular injury. It is suggested that TIPE1 may be involved in the pathogenesis of acute kidney injury.     

Complement C5a receptor antagonist attenuates multiple organ injury in a model of ruptured abdominal aortic aneurysm

OBJECTIVE: Abdominal aortic aneurysm (AAA) rupture is associated with a systemic inflammatory response syndrome, characterized by increased microvascular permeability and neutrophil sequestration, leading to multiorgan dysfunction. We examined the role of a novel complement factor 5a (C5aR) receptor antagonist, the cyclic peptide AcF-(OpdChaWR), in attenuation of pathologic complement activation and tissue injury in a model of AAA rupture. METHODS: Anesthetized rats were randomized to sham (control) or shock and clamp (s+c) groups. Animals in the s+c group underwent 1 hour of hemorrhagic shock (mean arterial blood pressure < or =50 mm Hg), followed by 45 minutes of supramesenteric aortic clamping, then 2 hours of resuscitated reperfusion. Animals in the s+c group were randomized to receive an intravenous bolus of C5aR antagonist at 1 mg/kg or saline solution control at the end of hemorrhagic shock. Intestinal and pulmonary permeability to iodine 125-labeled albumin was measured as an indicator of microvascular permeability. Tissue myeloperoxidase activity, proinflammatory cytokine tissue necrosis factor-alpha (TNF-alpha) protein and mRNA, and C5aR mRNA levels were measured as indicators of neutrophil sequestration and inflammatory signaling, respectively. RESULTS: Lung permeability index was significantly increased in the s+c group compared with the sham group (4.43 +/- 0.96 vs 1.30 +/- 0.17; P <.01), and prevented with treatment with C5aR antagonist (1.74 +/- 0.50; P <.03). Lung myeloperoxidase activity was significantly increased in the the s+c group compared with the sham group (2.41 +/- 0.34 U/mg vs 1.03 +/- 0.29 U/mg; P <.009), and significantly attenuated with treatment with C5aR antagonist (1.11 +/- 0.09 U/mg; P <.006). Lung TNF-alpha protein levels were significantly elevated in both s+c groups, whereas lung TNF-alpha mRNA expression was significantly downregulated in both s+c groups compared with the sham group. Intestinal permeability index was significantly increased in animals in the s+c groups during reperfusion, compared with sham (P <.001), which was attenuated in early reperfusion with treatment with C5a receptor antagonist. Data represent mean +/- SEM, group comparisons with analysis of variance and post hoc Scheffé test. CONCLUSIONS: These results indicate that a potent antagonist of C5a receptor protects the rat intestine and lung from neutrophil-associated injury in a model of AAA rupture. These data suggest that complement-mediated inflammation can be modulated at the C5a receptor level, independent of proinflammatory TNF-alpha production, and prevent acute local and remote organ injury.     

HLA-G与肾移植术后急性排斥反应和巨细胞病毒活动性感染的相关性研究

目的 探讨HLA-G的表达水平与肾移植术后急性排斥反应(AR)和巨细胞病毒(CMV)活动性感染的相关性.方法 根据术后是否发生AR或CMV活动性感染,将132例初次肾移植受者分为肾功能稳定组、AR组和CMV组.另选择41例健康供者作为对照组.采用流式细胞术、酶联免疫吸附试验、蛋白质印迹法以及实时定量聚合酶链法检测各组HLA-G及其mRNA的表达,并采用免疫组织化学法观察移植肾组织中HLA-G的表达.结果 肾移植前后各组膜结合型HLA-G1 (mHLA-G1)的表达均处于较低水平,仅术后CMV组mHLA-G1+的中性粒细胞出现显著升高(P＜0.05).术前可溶性HLA-G5(sHLA-G5)的表达水平肾功能稳定组显著高于对照组(P＜0.05);术后sHLA-G5的表达水平CMV组显著高于肾功能稳定组(P＜0.05),而肾功能稳定组均高于对照组和AR组(P＜0.05),AR组与对照组的差异无统计学意义(P＞0.05).术后CMV组sHLA-G5 mRNA的表达水平最高(P＜0.05),肾功能稳定组次之,对照组和AR组均较低.21例AR组移植肾组织活检样本中,17例HLA-G表达呈阴性,3例呈阳性,1例呈弱阳性;9例CMV组移植肾组织活检样本的HLA-G表达均为阳性.132例受者中,28例CMV感染者的AR发生率为7.1％(2/28),104例非CMV感染者的AR发生率为25.0％(26/104),二者间AR发生率的差异有统计学意义(P＜0.05).结论 sHLA-G5可作为预测AR和CMV感染的生物标志分子;CMV感染和AR与受者体内的免疫平衡状况相关.     

JAK2-STAT3 pathway regulates the expression of complement factor B in autosomal dominant polycystic kidney disease

Objective To investigate the role of JAK2-STAT3 pathway in the expression of complement factor B (CFB) in autosomal dominant polycystic kidney disease (ADPKD). Methods Renal tissue samples of patients with ADPKD after nephrectomy were collected. Normal renal tissue samples as control were taken from patients after radical nephrectomy. Renal tissue samples of Han: SPRD Cy/+ rats (ADPKD model) and wild-type Han: SPRD +/+ rats were also collected at 4, 8, 16 week. Han:SPRD Cy/+ rat renal tubular epithelial cells (16 w) were primarily cultured in vitro, then stimulated with the JAK2 inhibitor (WP1066) and STAT3 inhibitor (pyrimethamine) for 24 h respectively. Western blotting was used to detect the expression of p-JAK2, JAK2, p-STAT3, STAT3, CFB protein. Results Compared with control group, the protein expressions of p-JAK2, p-STAT3, STAT3, CFB significantly increased in the renal tissue of ADPKD patients (all P＜0.05). The protein expressions of p-JAK2, JAK2, p-STAT3, STAT3 and CFB also significantly increased in the renal tissue of Cy/+ rats compared with wild-type rats (all P＜0.01). When the Cy/+ renal tubular epithelial cells were treated with WP1066, the expressions of p-JAK2, p-STAT3, CFB were suppressed (P＜0.05) and the degree of inhibition was correlated with the WP1066 dose. Pyrimethamine inhibited the protein expressions of p-STAT3 and CFB in the tubular epithelial cells of Cy/+ rats (all P＜0.05) and the degree of inhibition was correlated with the pyrimethamine dose. Conclusions The JAK2-STAT3 pathway is abnormally activated in ADPKD and increases the protein expression of CFB. CFB protein level is correlated with the progress of ADPKD, suggesting that it may take part in the growth and development of ADPKD vesicles.     

Ubiquitination and endoplasmic reticulum stress of renal intrinsic cells in hyperglycemia

Objective To investigate the effects of hyperglycemia on ubiquitination and endoplasmic reticulum stress in renal intrinsic cells (podocytes and proximal tubular epithelial cells) and its role in pathogenesis of diabetic nephropathy. Methods Diabetic mice were induced by streptozotocin injection. After 16 weeks of hyperglycemia, immunofluorescence was used to detect the expressions of ubiquitination and glucose-regulating protein 94 (GRP94) in renal cortex and medulla area of kidney sections. Primary mouse podocyte and proximal tubular epithelial cells were isolated by flow cytometry, and exposed to 30 mmol/L glucose for indicated time (1 d, 3 d and 7 d). Their ubiquitination and GRP94 expressions were evaluated by Western blotting. Results Diabetic mice presented microalbuminuria and slightly widened mesangium was found in glomerular area. Ubiquitinated proteins, mainly localized in podocytes and tubular epithelial cells, exhibited an apparently higher expression in diabetic mice than control mice (all P＜0.05). Hyperglycemia promoted the ubiquitination in a time-dependent manner. Compared with their normal cells, primary mouse podocyte and primary tubular epithilial cells treated with high glucose for 3 d and 7 d showed increased ubiquitinated protein (all P＜0.05). GRP94 was interspersed in podocytes and proximal tubular epithelial cells. Expression of GRP94 was significantly increased in glomerular area of diabetic mice and podocyte with 3 and 7 day-high glucose as compared with those in their control groups (all P＜0.05). GRP94 expression had no significant change in tubular area and tubular epithilial cells treated with high glucose. Conclusions Hyperglycemia may lead to accumulation of ubiquitinated proteins in intrinsic kidney cells. The imbalance of protein homeostasis in podocyte may contribute to podocyte injury during the onset of diabetic nephropathy.     

Interleukin 10 knockout increases renal fibrosis of ischemia-reperfusion injury model mice

Objective To study the effect of interleukin (IL)-10 knockout (IL-10-/-) on renal repair after renal ischemia-reperfusion injury in mice. Methods Eighteen IL-10-/- mice (KO) aged 8-10 weeks and 18 C57BL/6 wild type mice (WT) aged 8-10 weeks were divided into control group (Sham) and renal ischemia-reperfusion injury (IRI) group. The renal tissue morphology change was observed by Hematoxylin and eosin (HE) staining and Masson staining. The expressions of IL-18, Ki67 and TGF-β1 were detected by immunohistochemistry. The expression of TGF-beta1 and IL-18 were detected by Western blotting. Results Compared with that in WT-IRI group, in KO-IRI group renal pathological damage was more severe, renal interstitial fibrosis was visible, Ki67 expression of renal tubular epithelial cells decreased distinctly (P＜0.01), the expression of TGF-beta1 increased significantly (P＜0.01). Conclusion Repair slows down significantly after kidney ischemia-reperfusion injury and fibrosis occurs gradually in IL-10-/- mice, eventually progressing to chronic kidney disease.