Role of apoptosis and Bcl-2/Bax in the development of tubulointerstitial fibrosis during experimental obstructive nephropathy

BACKGROUND/AIMS: To examine the role of apoptosis in experimental unilateral ureteral obstruction (UUO). METHODS: Rat kidneys were examined 3, 7 and 11 days following UUO or sham operation (SO). Tissue was immunohistochemically stained for alpha-smooth muscle actin (alpha-SMA), proliferating cell nuclear antigen (PCNA), Bcl-2 and Bax proteins. Apoptotic analysis was carried out in kidney sections using in situ end labelling of endonuclease cleaved DNA. RESULTS: The relative volume (Vv) of cortical interstitium and interstitial alpha-SMA increased progressively following UUO. ED1-positive monocytes/macrophages peaked at day 7 and significantly decreased at day 11. PCNA-positive cells in tubulointerstitium were significantly increased on day 3. Staining returned to the level of the SO group by day 11, meanwhile those in the interstitium remained much higher than baseline. TUNEL-positive cells were persistently raised following UUO. Transient tubular cell proliferation seemed unable to counteract the apoptosis since tubular atrophy was apparently present by day 11 of UUO. However, interstitial cell proliferation was high enough to overwhelm apoptosis, particularly with respect ot myofibroblasts, since alpha-SMA immunostaining and Vv remained elevated. The ratio of the number of PCNA-positive cells to apoptotic cells formed a predictive pattern for the staining score of interstitial alpha-SMA (R(2) = 47.23%, p < 0.05) and Vv (R(2) = 49.93%, p < 0.05). Tubular Bcl-2 immunostaining peaked on day 3, and then gradually decreased to baseline by day 11. The expression of Bax protein was inhibited on day 3 when compared with that of the SO group, but increased with time following UUO. CONCLUSION: These findings suggest an important role for apoptosis and its regulatory proteins in the processes of tubular atrophy and fibrogenesis following UUO.     

PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction

BACKGROUND: Progressive renal disease is characterized by the induction of plasminogen activator inhibitor-1 (PAI-1), suggesting that impaired activity of the renal plasmin cascade may play a role in renal fibrosis. METHODS: To test this hypothesis, the severity of renal fibrosis caused by unilateral ureteral obstruction (UUO) was compared in PAI-1 wild-type (+/+) and PAI-1 deficient (-/-) mice. The extent of interstitial inflammation and fibrosis, renal plasminogen activator and plasmin activity, and renal expression of profibrotic genes was evaluated after 3, 7, and 14 days of UUO. RESULTS: Renal PAI-1 mRNA levels increased 8- to 16-fold in the +/+ mice after UUO surgery, and PAI-1 protein was detected in kidney homogenates. Interstitial fibrosis was significantly attenuated in -/- mice compared with +/+ mice at day 7 and day 14, based on the interstitial area stained with picrosirius red and total kidney collagen content. However, neither the mean renal plasminogen activator nor plasmin activities were increased in -/- mice compared with +/+ mice. The number of interstitial macrophages were significantly lower in the -/- mice three and seven days after UUO; interstitial myofibroblasts were significantly fewer at three days. At the same time points, this altered interstitial cellularity was associated with a significant reduction in renal mRNA levels for transforming growth factor-beta and procollagens alpha 1(I) and alpha 1(III). CONCLUSIONS: These studies establish an important fibrogenic role for PAI-1 in the renal fibrogenic response. The results demonstrate that one important fibrosis-promoting function of PAI-1 is its role in the recruitment of fibrosis-inducing cells, including myofibroblasts and macrophages.     

Urokinase receptor modulates cellular and angiogenic responses in obstructive nephropathy

Interstitial cells have been implicated in the pathogenesis of renal fibrosis. Given that the urokinase receptor (uPAR) is known to play a role in cell adhesion, migration, and angiogenesis, the present study was designed to evaluate the role of uPAR in the regulation of the phenotypic composition of interstitial cells (macrophages, myofibroblasts, capillaries) in response to chronic renal injury. Groups of uPAR wild-type (+/+) and knockout (-/-) mice were investigated between 3 and 14 d after unilateral ureteral obstruction (UUO) or sham surgery (n = 8 mice per group). The density of F4/80+ interstitial macrophages (Mphi) was significantly lower in the -/- mice (3.3 +/- 0.4 versus 6.9 +/- 1.7% area at day 3 UUO; 10.8 +/- 1.6 versus 15.7 +/- 1.0% at day 14 UUO; -/- versus +/+). In contrast, in the -/- mice there were significantly more alpha smooth muscle actin (alphaSMA)-positive cells (12.9 +/- 3.2 versus 7.8 +/- 1.5% area at day 3 UUO; 21.0 +/- 4.7 versus 9.7 +/- 1.9% at day 14 UUO) and CD34-positive endothelial cells (8.4 +/- 1.9 versus 4.0 +/- 1.1% area at day 14 UUO). These differences were associated with significantly more interstitial fibrosis in the -/- mice based on Sirius red staining (4.6 +/- 0.9 versus 2.3 +/- 0.9% area at 14 d UUO). Absence of the uPAR scavenger receptor was associated with significantly greater accumulation of plasminogen activator inhibitor-1 protein (PAI-1) (20.5 +/- 3.5 versus 9.1 +/- 2.9% area, day 14 UUO) and vitronectin protein (2.4 +/- 1.1 versus 0.9 +/- 0.4% area, day 14 UUO). By immunostaining alphaSMA+ cells, CD34+ cells, vitronectin and PAI-1 co-localized to the same tubulointerstitial area. The number of apoptotic cells increased in response to UUO but was significantly higher in the -/- mice (2.0 +/- 0.2 versus 1.2 +/- 0.2 per 100 tubulointerstitial cells, day 14 UUO) while the number of proliferating cells was significantly lower in the uPAR-/- mice. These data suggest that uPAR deficiency suppresses renal Mphi recruitment, but the absence of this scavenger receptor actually accentuates the fibrogenic response, likely due in part to the delayed clearance of angiogenic/profibrotic molecules such as PAI-1 and decreased receptor-associated uPA activity.     

Chemokine receptor CCR1 but not CCR5 mediates leukocyte recruitment and subsequent renal fibrosis after unilateral ureteral obstruction

As chemokine receptor CCR1 and CCR5 expression on circulating leukocytes is thought to contribute to leukocyte recruitment during renal fibrosis, the authors examined the effects of unilateral ureteral obstruction (UUO) in mice deficient for CCR1 or CCR5. Analysis of UUO kidneys from CCR1-deficient mice revealed a reduction of interstitial macrophages and lymphocytes (35% and 55%, respectively) compared with wild-type controls. CCR1-deficient mice had reduced CCR5 mRNA levels in UUO kidneys, which correlated with a reduction of CCR5+ T cell infiltrate as determined by flow cytometry. Interstitial fibroblasts, renal TGF-beta1 mRNA expression, interstitial volume, and collagen I deposits were all significantly reduced in CCR1-deficient mice. In contrast, renal leukocytes and fibrosis were unaffected in CCR5-deficient mice with UUO. However, if treated with the CCR1 antagonist BX471, CCR5-deficient mice showed a similar reduction of renal leukocytes and fibrosis as CCR1-deficient mice. To determine the underlying mechanism labeled macrophages and T cells isolated from either wild-type, CCR1-deficient, or CCR5-deficient mice were injected into wild-type mice with UUO. Three hours later, renal cell recruitment was reduced for CCR1-deficient cells or cells pretreated with BX471 compared with CCR5-deficient or wild-type cells. Thus, CCR1 but not CCR5 is required for leukocyte recruitment and fibrosis after UUO in mice. Therefore, CCR1 is a promising target for therapeutic intervention in leukocyte-mediated fibrotic tissue injury, e.g. progressive renal fibrosis.     

Progressive renal fibrosis in murine polycystic kidney disease: an immunohistochemical observation

BACKGROUND: The appearance of interstitial fibrosis in polycystic kidneys is emblematic of progressive disease. Matrix forming this scar tissue is derived from local renal cells in response to cystogenesis. We investigated the phenotype of collagen-producing cells in the cystic kidneys of DBA/2-pcy mice to better characterize the spectrum of interstitial cells associated with renal fibrogenesis. METHODS: The extent of interstitial fibrosis and the number of fibroblasts in cystic kidneys were first quantitated over time using computer-assisted image analysis. Subsequently, antisera to four cell protein markers were studied by coexpression immunohistochemistry during progression of fibrosis using confocal microscopy. The antisera included fibroblast-specific protein 1 (FSP1) for fibroblast phenotype, alpha-smooth muscle actin (alpha-SMA) for contractile phenotype, vimentin (VIM) for mesenchymal phenotype, and heat shock protein 47 (HSP47) for interstitial collagen-producing phenotype. RESULTS: Interstitial fibrosis in cystic kidneys gradually increased throughout the 30-week observation period of our study. With progression of cystogenesis, most of the tubules in pcy mice either dilated or disappeared with time. FSP1+ fibroblasts were distributed sparsely throughout the renal interstitium of young pcy and wild-type mice. Their number increased in the widening fibrotic septa by 18 weeks of age and persisted through 30 weeks of the study interval. Some epithelia among remnant tubules trapped within fibrotic septa around adjacent cysts also acquired the phenotype of FSP1+, HSP47+ collagen-producing fibroblasts, suggesting a possible role for epithelial-mesenchymal transformation (EMT) in this process. Most FSP1+ fibroblasts were alpha-SMA-, but HSP47+, suggesting they were producing collagen proteins for the extracellular matrix. alpha-SMA+, FSP1-, HSP47+ or HSP47- cells were also observed, and the latter tended to distribute independently in a linear pattern, reminiscent of vasculature adjacent to forming cysts. VIM+ expression was not observed in alpha-SMA+ cells. CONCLUSIONS: Many nonoverlapping as well as fewer overlapping populations of FSP1+ and alpha-SMA+ cells shared in the collagen expression associated with progressive fibrogenesis in pcy mice undergoing cystogenesis. Some FSP1+ fibroblasts are likely derived from tubular epithelium undergoing EMT, while alphaSMA+, VIM- cells probably represent vascular smooth muscle cells or pericytes surviving vessel attenuation during the chaos of fibrogenesis. Importantly, not all interstitial cells producing collagens are alpha-SMA+.     

Attenuation of ureteral obstruction-induced renal injury by polyenylphosphatidylcholine

BACKGROUND: The cytoprotective, antioxidant and antifibrotic effects of polyenylphosphatidylcholine (lecithin, PPC) have been demonstrated both experimentally and clinically. The present study investigated whether PPC treatment has any beneficial effect on renal injury in unilateral partial ureteral obstruction (UUO) in rats. METHODS: Forty Wistar-Albino rats were split into three groups (sham-operated controls, untreated and treated rats). Rats of the untreated and treated groups (n = 15) underwent UUO with two-thirds of the left ureter embedded in the psoas muscle. In group 3, PPC was given orally at a dose of 100 mg/day for 30 days. At the end of the 30th day of the experimental period, obstructed kidneys and blood samples were harvested. To investigate the therapeutic efficacy of PPC treatment in UUO kidneys, oxidant and antioxidant enzyme levels, lipid peroxidation, proinflammatory cytokines (interleukin-1, interleukin-6, tumor necrosis factor alpha), transforming growth factor beta-1 (TGFbeta-1), alpha smooth muscle actin (alpha-SMA) and nuclear factor kappa beta (NF-kappabeta) expression, leukocyte infiltration (ED1, ED2, CD4 and CD8 immunohistochemistry), and tubulointerstitial damage in the obstructed kidneys were studied. RESULTS: Oxidative stress, neutrophil infiltration, release of cytotoxic mediators, TGFbeta-1 levels, tubulointerstitial damage, alpha-SMA and NF-KB expressions in kidney tissue were significantly increased in the UUO rats. PPC treatment attenuated oxidative stress, leukocyte infiltration, cytotoxic mediator, and TGFbeta-1 levels and also decreased expressions of alpha-SMA and NF-kappabeta. It was associated with decreased tubulointerstitial damage, compared with UUO alone. CONCLUSIONS: These results indicate that PPC treatment protects against UUO-induced renal injury in rats possibly through its antioxidant, anti-inflammatory and antifibrotic actions.     

Multifunctionality of PAI-1 in fibrogenesis: evidence from obstructive nephropathy in PAI-1-overexpressing mice

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of chronic kidney disease based on its up-regulated expression and on the beneficial effects of PAI-1 inhibition or depletion in experimental models. PAI-1 is a multifunctional protein and the mechanisms that account for its profibrotic effects have not been fully elucidated. METHODS: The present study was designed to investigate PAI-1-dependent fibrogenic pathways by comparing the unilateral ureteral obstruction model (UUO) (days 3, 7, and 14) in PAI-1-overexpressing mice (PAI-1 tg) to wild-type mice, both on a C57BL6 background. RESULTS: Following UUO, total kidney PAI-1 mRNA and/or protein levels were significantly higher in the PAI-1 tg mice (N= 6 to 8/group) and fibrosis severity was significantly worse (days 3, 7, and 14), measured both as Sirius red-positive interstitial area (e.g., 10 +/- 3.2% vs. 4.5 +/- 1.0%) (day 14) and total kidney collagen (e.g., 11.1 +/- 1.7 vs. 6.2 +/- 1.3 microg/mg) (day 14). By day 14, the expression of two normal tubular proteins, E-cadherin and Ksp-cadherin, were significantly lower in the PAI-1 tg mice (3.2 +/- 0.5% vs. 11.7 +/- 5.9% and 2.6 +/- 1.6) vs. 6.2 +/- 0.8%, respectively), implying more extensive tubular damage. At least four fibrogenic pathways were differentially expressed in the PAI-1 tg mice. First, interstitial macrophage recruitment was more intense (P < 0.05 days 3 and 14). Second, interstitial myofibroblast density was greater (P < 0.05 days 3 and 7) despite similar numbers of proliferating tubulointerstitial cells. Third, transforming growth factor-beta1 (TGF-beta1) and collagen I mRNA were significantly higher. Finally, urokinase activity was significantly lower (P < 0.05 days 7 and 14) despite similar mRNA levels. Gene microarray studies documented that that the deletion of this single profibrotic gene had far-reaching consequences on renal cellular responses to chronic injury. CONCLUSION: These data provide further evidence that PAI-1 is directly involved in interstitial fibrosis and tubular damage via two primary overlapping mechanisms: early effects on interstitial cell recruitment and late effects associated with decreased urokinase activity.     

干细胞因子联合粒细胞集落刺激因子动员骨髓干细胞促进单侧输尿管梗阻大鼠肾脏损伤的修复

目的 研究干细胞因子（SCF）联合粒细胞集落刺激因子（G-CSF）动员单侧输尿管梗阻（UUO）大鼠骨髓干细胞对肾间质中微血管、纤维化程度和肾功能的影响,并探讨其对微血管影响的可能机制。 方法 128只大鼠按数字随机法分为假手术组（Sham组）、SCF联合G-CSF动员组（SCF-G组）、UUO组、UUO+SCF-G组。于实验第 5、14、21、28天每组各随机抽取8只处死,检测Scr、肾间质CD34阳性表达细胞数目和Ⅷ因子阳性表达细胞数目、肾间质纤维化和间质病理损害积分、肾皮质血管内皮生长因子（VEGF）mRNA和血小板反应蛋白1（TSP-1）mRNA的表达。 结果 (1)UUO组2周时可见到肾间质纤维化伴肾小管周微血管的丢失。（2）UUO+SCF-G组肾间质干细胞归巢数目明显高于UUO组和Sham组（P < 0.05）。（3）UUO+SCF-G组肾小管周微血管指数减少出现的时间晚于UUO组（P < 0.05）。（4）第14、21、28天UUO+SCF-G组间质化纤维程度和肾小管损伤程度均轻于UUO组（P < 0.05）。（5）UUO+SCF-G组术后VEGF mRNA表达下调出现的时间晚于UUO组,且表达均高于同期UUO组 （P < 0.05)。（6）UUO+SCF-G组术后TSP-1 mRNA表达增高出现的时间晚于UUO组,且表达均低于同期UUO组（P < 0.05）。（7）在UUO组和UUO+SCF-G组中,肾小管周微血管指数与Scr、间质纤维化积分和肾小管间质病理积分均呈负相关;肾皮质VEGF mRNA表达与肾小管周微血管指数呈正相关;肾皮质TSP-1 mRNA表达与肾小管周微血管指数呈负相关。 结论 （1）UUO大鼠存在肾小管周微血管丢失,并与肾间质纤维化及间质病理损伤相关。（2）联合应用SCF和G-CSF动员骨髓干细胞可以归巢至受损的肾脏,有助于减少肾小管周微血管丢失,并进而减轻肾间质纤维化和间质损害,保护肾功能。（3）联合应用SCF和G-CSF可以上调肾皮质VEGF mRNA水平和下调TSP-1 mRNA水平,这可能是其促进内皮细胞修复及保护肾间质微血管损伤的机制之一。     

Transforming growth factor-beta induces renal epithelial jagged-1 expression in fibrotic disease

For elucidation of the mechanisms by which growth factors and cytokines affect renal epithelial cells, gene array analysis of renal cells cultured in the presence of transforming growth factor-beta1 (TGF-beta1) was performed. Many genes that were not previously considered to be involved in renal cell biologic processes were affected, one of which was jagged-1. The jagged ligand/notch receptor family controls the formation of boundaries between groups of cells and regulates cell fates. On the basis of the array analysis, jagged-1 expression was further evaluated in cultured cells and in C57BL/6 mice with a model of unilateral ureteral obstruction (UUO). Recombinant human TGF-beta1 increased jagged-1 mRNA levels at concentrations between 10(-11) and 10(-10) M. There was a commensurate increase in jagged-1 protein levels, as assessed by Western blotting. The expression of jagged-1 mRNA and protein was observed to be significantly increased in the kidneys of C57BL/6 mice with obstructed ureters, compared with the contralateral kidneys, at 7 and 14 d of UUO. Immunohistochemical analyses demonstrated jagged-1 expression in distal tubules of kidneys from normal mice or contralateral kidneys from mice with UUO. Jagged-1 protein expression was increased in tubules not yet in apparent atrophy in the kidneys with an obstructed ureter. Jagged-1 expression was significantly increased in the kidneys of normal mice treated with TGF-beta1 and was decreased in the kidneys of mice with UUO treated with a TGF-beta receptor II-Fc chimera. These results suggest that jagged-1 is expressed in normal kidneys and that this expression is upregulated during renal disease, in a TGF-beta-dependent manner.     

环加氧酶在梗阻性肾病大鼠肾脏的表达及莫比可的作用机制研究

目的 观察环加氧酶(COX)在梗阻性肾病大鼠肾脏的表达,比较选择性(COX-2抑制剂莫比可与消炎痛对COX、纤溶酶原激活物抑制物1(PAI-1)和基质金属蛋白酶组织抑制物-1(TIMP-1)基因转录的影响。方法 将输尿管梗阻大鼠随机分成不用药组(U)、消炎痛组(I)和莫比可组(M),设假手术组(C)为对照。4周后应用逆转录-聚合酶链反应(RT-PCR)检测COX-1、COX-2、PAI-1及TIMP-1的mRNA表达,免疫组织化学检测COX-1和COX-2的蛋白水平,并采用放射免疫法测定术侧肾脏残余尿液的血栓烷素B2(TXB2)浓度。结果 COX-1的mRNA和蛋白水平在4组间差异无显著性意义。U组大鼠尿TXB2浓度急剧上升,术肾PAI-1和TIMP-1的mRNA表达明显上调,COX-2的基因表达和蛋白质合成大幅增加。莫比可能够显著抑制上述基因的转录(P<0.05),而I组的数值则与U组相仿。M组和I组的尿TXB2浓度明显下降,尤以M组为著。结论 COX-2参与了UUO发生、发展的病理过程。选择性COX-2抑制剂能够从抑制COX-2活性、下调COX-2、PAI-1及TIMP-1的基因转录等多方面发挥其积极的治疗作用。     

Proximal tubular cells promote fibrogenesis by TGF-beta1-mediated induction of peritubular myofibroblasts

BACKGROUND: In proteinuric nephropathies with increasingly severe defects of the glomerular filtering barrier, interstitial fibrogenesis is a major effector of scarring. An early event in this process is the peritubular accumulation of myofibroblasts that express alpha-smooth muscle actin (alpha-SMA) and contribute to abnormal matrix production. Common trigger factors are poorly understood. Enhanced protein trafficking may play a role by up-regulating inflammatory and fibrogenic genes in proximal tubular cells. METHODS: The remnant kidney model in rats was used to (1) analyze interactions between activated proximal tubular cells, peritubular cells expressing the myofibroblast marker, and inflammatory cells at time intervals (days 7, 14, and 30) after surgery, and (2) evaluate the effects of angiotensin-converting enzyme inhibitor (ACEi) on protein trafficking, fibrogenic signaling, and alpha-SMA expression. RESULTS: Abnormal uptake of ultrafiltered proteins by proximal tubular cells (IgG staining) occurred at an early stage (day 7) and was subsequently associated with macrophage and alpha-SMA+ cell accumulation into the peritubular interstitium. alpha-SMA+ cells clustered with macrophages into the interstitium. These changes were associated with appearance of transforming growth factor-beta1 (TGF-beta1) mRNA in proximal tubular cells and in the infiltrating cells with time. At day 30, focal alpha-SMA staining also was found in the tubular cells and in peritubular endothelial cells on semithin ultracryosections. ACEi prevented both proteinuria and abnormal protein accumulation in tubular cells, as well as the inflammatory and fibrogenic reaction with peritubular alpha-SMA expression. CONCLUSIONS: Profibrogenic signaling from both proximal tubular cells on challenge with filtered protein and inflammatory cells is implicated as a key candidate trigger of progressive tubulointerstitial injury.     

TIMP-1 deficiency does not attenuate interstitial fibrosis in obstructive nephropathy

Progressive renal disease as a result of renal fibrosis is caused in part by an impairment of the proteolytic machinery that normally regulates matrix turnover. The goal of the present study was to determine whether genetic deficiency of tissue inhibitor of metalloproteinases-1 (TIMP-1) could attenuate interstitial fibrosis caused by unilateral ureteral obstruction (UUO). Groups of wild-type (Timp-1) mice and TIMP-1-deficient (timp-1) mice were killed after 3 and 14 d of UUO or sham operation. Timp-1 mRNA levels were significantly increased 37- and 19-fold in the wild-type mice 3 and 14 d, respectively, after UUO operation. Matrix metalloproteinase-9 (MMP-9) activity fell in all UUO groups but remained significantly higher in the timp-1 group compared with the Timp-1 group. The degree of interstitial fibrosis (kidney collagen content and percentage of tubulointerstitial area stained with picrosirius red and collagen III) was significantly increased 14 d after UUO operation, but there was no difference between the Timp-1 and timp-1 groups. Many features of the fibrogenic response were similar between the Timp-1 and timp-1 groups, including the number of myofibroblasts and the induction of genes encoding procollagen III, fibronectin, and transforming growth factor-beta. After UUO operation, renal mRNA levels for Timp-3 and plasminogen activator inhibitor-1 were significantly higher in the TIMP-1-deficient mice. The results of this study show that elimination of TIMP-1 alone does not alter the severity of interstitial fibrosis. These findings may be due to compensation by other protease inhibitors such as TIMP-2, TIMP-3, and/or plasminogen activator inhibitor-1 or to the possibility that inhibition of intrinsic MMP activity does not constitute a profibrogenic event in the kidney.     

血小板反应蛋白-1及其受体CD47在肾间质纤维化血管病变中的表达

目的观察血小板反应蛋白-1(TSP-1)及其受体CD47在单侧输尿管梗阻(UUO)大鼠模型中的表达,探讨TSP-1-CD47在肾周毛细血管(PTC)病变中的作用。 方法将60只SD大鼠随机分为2组：UUO组45只和假手术组(SOR) 15只。UUO组随机选取15只分别于术后3 d、7 d、14 d处死;SOR组随机选取5只于相同时间点处死。检测24 h尿蛋白定量,采用全自动生化分析仪检测各组大鼠的肾功能,通过Masson染色观察肾间质病理改变;免疫荧光双染色观察TSP-1及其受体CD47在UUO模型中是否存在共表达。Western印迹检测毛细血管病变指标CD34和血管内皮生长因子(VEGF)及TSP-1的表达;多组间均数比较使用方差分析;TSP-1与CD34、VEGF的相关性进行Pearson相关分析。 结果与SOR组相比,UUO各组大鼠血清肌酐、尿素氮、24 h尿蛋白定量随梗阻时间延长无明显变化,但肾间质纤维化逐渐加重,肾小管损伤评分增加。免疫荧光双染色显示TSP-1及其受体CD47在肾小管间质存在共表达。Western印迹检测显示随UUO时间延长,TSP-1蛋白水平逐渐增加,而CD34及VEGF蛋白表达随梗阻时间延长逐渐下降。相关分析显示TSP-1蛋白水平与CD34及VEGF的表达呈负相关(r＝－0.931, P<0.01;r＝－0.953, P<0.01)。 结论TSP-1及其受体CD47在UUO大鼠模型肾间质存在共表达,且与PTC呈负相关。TSP-1可能通过CD47参与UUO肾脏PTC病变的发生发展。     

Role of atrophic changes in proximal tubular cells in the peritubular deposition of type IV collagen in a rat renal ablation model.

BACKGROUND: Tubular atrophy, dilation and interstitial fibrosis are common in tubulointerstitial lesions, but the precise roles and inter-relationships of these components in the development of interstitial lesions have not been determined. This study focused on the origin and roles of atrophic tubules in the peritubular deposition of type IV collagen in a rat renal ablation model. METHODS: Male Wistar rats underwent 5/6 nephrectomy or sham operation, and then were sacrificed at 4, 8 or 12 weeks, their remaining kidneys removed for histological and immuno-histochemical studies as well as in situ hybridization for type IV collagen mRNA. RESULTS: Immuno-histochemistry demonstrated the positive staining of atrophic tubules to vimentin, platelet-derived growth factor-B chain (PDGF) and heat shock protein 47 (HSP47). Cells positive to one or more of PDGF receptor beta, alpha-smooth muscle actin (alpha-SMA), and HSP47 accumulated around atrophic tubules. Type IV collagen was also increased in the proximity of the atrophic tubules. These intimate relationships were more clearly demonstrated in 'mosaic tubules', which are composed of both intact and atrophic proximal tubular epithelial cells, and which had a mixed pattern of staining with vimentin, PDGF and HSP47. The interstitial cells positive to alpha-SMA or HSP47, or both, were in close contact with atrophic but not with intact epithelial cells. Type IV collagen was exclusively deposited between atrophic tubules and HSP47-positive interstitial cells. In situ hybridization of type IV collagen mRNA demonstrated predominant expression in atrophic tubular epithelial cells, but not in surrounding interstitial cells. CONCLUSIONS: These findings suggest that atrophic proximal tubular cells are active in the development of collagen deposition in the peritubular space, i.e. in this model type IV collagen in the interstitial fibrotic area may be produced mainly by atrophic proximal tubules.     

NGAL、MMP-9及TIMP-1在单侧输尿管梗阻大鼠肾组织中的表达

目的：动态观测单侧输尿管梗阻大鼠肾组织中中性粒细胞相关脂质运载蛋白（NGAL）、基质金属蛋白酶-9（MMP-9）及金属蛋白酶组织抑制因子（TIMP—1）的表达变化,探讨NGAL分子在肾小管间质纤维化发展中的作用机制。方法：将48只雄性SD大鼠随机分为UUO模型组及假手术（SOR）组,每组24只,UUO组行左侧输尿管梗阻术,SOR组仅游离左侧输尿管不结扎。术后第1、4、7、14天分别处死每组6只大鼠,取左肾行HE＆Masson染色,并用免疫组化EI,iVision‘M1plus法检测肾组织NGAL、MMP-9及TIMP-1的表达。结果：HE、Masson染色切片示：UUO组间质损伤指数在术后各时间点高于SOR组（P〈0．05）。免疫组化结果显示：UUO组术后各时间点NGAL表达高于SOR组（P〈0．05）,与肾小管损伤指数呈明显负相关;MMP-9在uu0术后早期（1～7天）有所上升,随着病程进展,7～14天表达有所下降;TIMP1在UUO术后表达逐渐增强;NGAI。与MMP-9／TIMP-1比值呈明显正相关。结论：NGAL在肾间质纤维化早期发挥负向调节作用,对调节MMP-9／TIMP-1的平衡亦发挥重要作用。     

Heme oxygenase-1 protects rat kidney from ureteral obstruction via an antiapoptotic pathway

This study examined the functional significance of heme oxygenase-1 (HO-1) expression on renal injury induced by ureteral obstruction in the rat kidney. Male Sprague-Dawley rats were divided into three groups, after which unilateral ureteral obstruction (UUO) was performed: untreated (group 1), treated with 30 mg/kg body wt hemin (group 2), and treated with 50 microg/kg body wt zinc (alpha) protoporphyrin eta (ZnPP) and 30 mg/kg hemin (group 3). After 7 and 14 d, histologic changes and the expression of HO-1, Bcl-2, Bad, TGF-beta, and cleaved caspase-3 were examined. Tubular lumens were dilated and epithelial cells were flattened on day 7 after UUO. Interstitial fibrosis and separation of the tubules were markedly increased on day 14. In contrast, the kidneys that were treated with hemin exhibited minimal interstitial fibrosis and flattening of epithelial cells on day 7 and fewer changes on day 14 than in the controls. However, treatment with ZnPP, an inhibitor of HO enzyme activity, eliminated the beneficial effect of hemin on interstitial fibrosis and tubular dilation. Increased HO-1 expression was associated with increased Bcl-2. In the ZnPP-treated rats, Bcl-2 signals were decreased compared with the hemin group. The level of proapoptotic Bad was not changed in any group. The positive cells for cleaved caspase-3 were significantly increased in renal tubular epithelial cells and tubulointerstitial cells in the obstructed rats, and hemin treatment decreased the caspase-3 activation. This study demonstrates that upregulation of HO-1 provides protection against renal injury that follows UUO. This effect is dependent on modulation of the antiapoptotic pathway by HO-1 expression.     

雌激素对单侧输尿管梗阻大鼠肾间质纤维化的保护作用

目的探讨雌激素对单侧输尿管梗阻（UUO）大鼠肾间质纤维化的作用。方法雌性SD大鼠30只，随机分为4组：Ⅰ组对照组；Ⅱ组生理雌激素组；Ⅲ组低雌激素组；Ⅳ组高雌激素组。UUO术后21d处死各组大鼠，光镜观察梗阻肾组织病理变化，并分别用免疫组化和RT-PCR方法检测各组肾组织α-平滑肌肌动蛋白（α-SMA）和金属蛋白酶1组织抑制剂（TIMP-1）的表达。结果低雌激素组间质纤维化病变最明显，高雌激素组病变显著减轻（P〈0．01）。与生理雌激素组相比，低雌激素组α—SMA和TIMP-1蛋白和基因的表达增加（P〈0．05）；高雌激素组上述物质表达则减少（P〈0．05）。结论雌激素可能通过抑制α-SMA和TIMP-1的表达进而减少细胞外基质的沉积而发挥肾保护作用。