Endoglin regulates renal ischaemia-reperfusion injury.

BACKGROUND: Renal ischaemia-reperfusion (I-R) can cause acute tubular necrosis and chronic renal deterioration. Endoglin, an accessory receptor for Transforming Growth Factor-beta1 (TGF-beta1), is expressed on activated endothelium during macrophage maturation and implicated in the control of fibrosis, angiogenesis and inflammation. METHODS: Endoglin expression was monitored over 14 days after renal I-R in rats. As endoglin-null mice are not viable, the role of endoglin in I-R was studied by comparing renal I-R injury in haploinsufficient mice (Eng(+/-)) and their wild-type littermates (Eng(+/+)). Renal function, morphology and molecular markers of acute renal injury and inflammation were compared. RESULTS: Endoglin mRNA up-regulation in the post-ischaemic kidneys of rats occurred at 12 h after I-R; endoglin protein levels were elevated throughout the study period. Expression was initially localized to the vascular endothelium, then extended to fibrotic and inflamed areas of the interstitium. Two days after I-R, plasma creatinine elevation and acute tubular necrosis were less marked in Eng(+/-) than in Eng(+/+) mice. Significant up-regulation of endoglin protein was found only in the post-ischaemic kidneys of Eng(+/+) mice and coincided with an increased mRNA expression of the TGF-beta1 and collagen IV (alpha1) chain genes. Significant increases in vascular cell adhesion molecule-1 (VCAM-1) and inducible nitric oxide synthase (iNOS) expression, nitrosative stress, myeloperoxidase activity and CD68 staining for macrophages were evident in post-ischaemic kidneys of Eng(+/+), but not Eng(+/-) mice, suggesting that impaired endothelial activation and macrophage maturation may account for the reduced injury in post-ischaemic kidneys of Eng(+/-) mice. CONCLUSIONS: Endoglin is up-regulated in the post-ischaemic kidney and endoglin-haploinsufficient mice are protected from renal I-R injury. Endoglin may play a primary role in promoting inflammatory responses following renal I-R.     

Enhancement of Crude Bone Morphogenetic Protein-Induced New Bone Formation and Normalization of Endochondral Ossification by Bisphosphonate Treatment in Osteoprotegerin-Deficient Mice

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor receptor family which plays a crucial role in negative regulation of osteoclastic bone resorption. We investigated both the quantity and quality of heterotopic new bone induced by crude bone morphogenetic protein (BMP) as a means of examining bone metabolism by bisphosphonate administration in OPG^−/− mice. Four weeks after implantation of crude BMP, the volume of heterotopic new bone in OPG^−/− mice without alendronate was significantly less than in wild-type (WT) mice. Alendronate treatment of OPG^−/− mice resulted in enhancement of the volume of heterotopic new bone. Histological findings revealed that WT mice showed normal bone formation with persistent cartilage that was interspersed with islands of bone. In contrast, the cartilage was replaced by trabecular bone and bone marrow adipocytes in OPG^−/− mice without alendronate. However, some cartilage was still present in OPG^−/− mice with alendronate compared to those without alendronate. All bone formation-related parameters and bone resorption-related parameters were significantly lower in OPG^−/− mice with alendronate than in those without alendronate. These findings suggest that in stimulated osteoclastogenesis without OPG, osteoinductive activity induced by crude BMP is inhibited and endochondral ossification induced by crude BMP is accelerated. On the other hand, alendronate treatment of OPG^−/− mice caused osteoinductive activity induced by crude BMP to increase and endochondral ossification induced by crude BMP to be decelerated. In conclusion, inhibition of stimulated osteoclastogenesis results in the enhancement of new bone formation and normalization of endochondral ossification.     

Skeletal effects of estrogen are mediated by opposing actions of classical and nonclassical estrogen receptor pathways.

ER alpha acts either through classical (ERE-mediated) or nonclassical (non-ERE) pathways. The generation of mice carrying a mutation that eliminates classical ER alpha signaling presents a unique opportunity to study the relative roles of these pathways in bone. This study defines the skeletal phenotype and responses to ovariectomy and estrogen replacement in these mice. INTRODUCTION: Estrogen receptor alpha (ER alpha) can act either through classical estrogen response elements (EREs) or through non-ERE (nonclassical) pathways. To unravel these in bone, we crossed mice heterozygous for a knock-in mutation abolishing ERE binding (nonclassical ER alpha knock-in [NERKI]) with heterozygote ER alpha knockout mice and studied the resulting female ER alpha(+/+), ER alpha(+/NERKI), and ER alpha(-/NERKI) mice. The only ER alpha present in ER alpha(-/NERKI) mice is incapable of activating EREs but can signal through nonclassical pathways, whereas ER alpha(+/NERKI) mice may have a less drastic alteration in the balance between classical and nonclassical estrogen signaling pathways. MATERIALS AND METHODS: BMD was measured using DXA and pQCT at 3 months of age (n = 46-48/genotype). The mice were randomly assigned to sham surgery, ovariectomy, ovariectomy + estradiol (0.25 microg/day), or ovariectomy + estradiol (1.0 microg/day; n = 10-12/group) and restudied 60 days later. RESULTS AND CONCLUSIONS: At 3 months of age, both the ER alpha(+/NERKI) and ER alpha(-/NERKI) mice had deficits in cortical, but not in trabecular, bone. Remarkably, changes in cortical bone after ovariectomy and estrogen replacement in ER alpha(-/NERKI) mice were the opposite of those in ER alpha(+/+) mice. Relative to sham mice, ovariectomized ER alpha(-/NERKI) mice gained more bone (not less, as in ER alpha(+/+) mice), and estrogen suppressed this increase (whereas augmenting it in ER alpha(+/+) mice). Estrogen also had opposite effects on bone formation and resorption parameters on endocortical surfaces in ER alpha(-/NERKI) versus ER alpha(+/+) mice. Collectively, these data show that alteration of the balance between classical and nonclassical ER alpha signaling pathways leads to deficits in cortical bone and also represent the first demonstration, in any tissue, that complete loss of classical ERE signaling can lead to paradoxical responses to estrogen. Our findings strongly support the hypothesis that there exists a balance between classical and nonclassical ER alpha signaling pathways, which, when altered, can result in a markedly aberrant response to estrogen.     

BMP‐9‐induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment

Heterotopic ossification (HO) is defined as the formation of bone inside soft tissue. Symptoms include joint stiffness, swelling, and pain. Apart from the inherited form, the common traumatic form generally occurs at sites of injury in damaged muscles and is often associated with brain injury. We investigated bone morphogenetic protein 9 (BMP‐9), which possesses a strong osteoinductive capacity, for its involvement in muscle HO physiopathology. We found that BMP‐9 had an osteoinductive influence on mouse muscle resident stromal cells by increasing their alkaline phosphatase activity and bone‐specific marker expression. Interestingly, BMP‐9 induced HO only in damaged muscle, whereas BMP‐2 promoted HO in skeletal muscle regardless of its state. The addition of the soluble form of the ALK1 protein (the BMP‐9 receptor) significantly inhibited the osteoinductive potential of BMP‐9 in cells and HO in damaged muscles. BMP‐9 thus should be considered a candidate for involvement in HO physiopathology, with its activity depending on the skeletal muscle microenvironment. © 2011 American Society for Bone and Mineral Research.     

Compound heterozygous loss of Ext1 and Ext2 is sufficient for formation of multiple exostoses in mouse ribs and long bones

Multiple Hereditary Exostoses (MHE) syndrome is caused by haploinsufficiency in Golgi-associated heparan sulfate polymerases EXT1 or EXT2 and is characterized by formation of exostoses next to growing long bones and other skeletal elements. Recent mouse studies have indicated that formation of stereotypic exostoses requires a complete loss of Ext expression, suggesting that a similar local loss of EXT function may underlie exostosis formation in patients. To further test this possibility and gain greater insights into pathogenic mechanisms, we created heterozygous Ext1(+/-) and compound Ext1(+/-)/Ext2(+/-) mice. Like Ext2(+/-) mice described previously (Stickens et al. Development 132:5055), Ext1(+/-) mice displayed rib-associated exostosis-like outgrowths only. However, compound heterozygous mice had nearly twice as many outgrowths and, more importantly, displayed stereotypic growth plate-like exostoses along their long bones. Ext1(+/-)Ext2(+/-) exostoses contained very low levels of immuno-detectable heparan sulfate, and Ext1(+/-)Ext2(+/-) chondrocytes, endothelial cells and fibroblasts in vitro produced shortened heparan sulfate chains compared to controls and responded less vigorously to exogenous factors such as FGF-18. We also found that rib outgrowths formed in Ext1(f/+)Col2Cre and Ext1(f/+)Dermo1Cre mice, suggesting that ectopic skeletal tissue can be induced by conditional Ext ablation in local chondrogenic and/or perichondrial cells. The study indicates that formation of stereotypic exostoses requires a significant, but not complete, loss of Ext expression and that exostosis incidence and phenotype are intimately sensitive to, and inversely related to, Ext expression. The data also indicate that the nature and organization of ectopic tissue may be influenced by site-specific anatomical cues and mechanisms.     

Twisted gastrulation, a bone morphogenetic protein agonist/antagonist, is not required for post-natal skeletal function

Twisted gastrulation (Tsg) is a secreted glycoprotein that binds bone morphogenetic proteins (BMP)-2 and -4 and can display both BMP agonist and antagonist functions. Tsg promotes BMP-mediated endochondral ossification, but its activity in adult bone is not known. We created tsg null mice and examined the consequences of the tsg deletion on the skeleton in vivo and on osteoblast function in vitro. Analysis of the skeletal phenotype of 4-week-old tsg null mice revealed a 40% decrease in trabecular bone volume, but osteoblast and osteoclast number, and bone formation and resorption were not affected. The phenotype was transient, and at 7 weeks of age tsg null mice were not different from control wild-type mice. The decreased trabecular bone is congruent with a defect in endochondral bone formation. In osteoblasts isolated from tsg null mice, tsg gene inactivation decreased the BMP-2 stimulatory effects on osteocalcin expression and alkaline phosphatase activity, indicating that in the bone microenvironment endogenous Tsg enhances BMP activity. Accordingly, tsg null cells displayed impaired BMP signaling. These results were confirmed by Tsg down-regulation in primary osteoblasts from wild-type mice using RNA interference. In conclusion, endogenous Tsg is required for normal BMP activity in osteoblastic cells in vitro, but it plays a minor role in the regulation of adult bone homeostasis in vivo.     

Increased levels of surgical adhesions in TGFbeta1 heterozygous mice.

Adhesion formation and fibrosis represent a major complication of surgical intervention. Reducing the morbidity associated with adhesions requires an understanding of the mechanisms underlying their formation. Since increased levels of transforming growth factor-beta1 (TGFbeta1) have been associated with inflammation and adhesion production, we investigated the requirement of TGFbeta1 in peritoneal adhesion formation utilizing mice carrying a targeted disruption of the TGFbeta1 allele. Mice that were either wild-type (+/+), containing two normal alleles of TGFbeta1, or heterozygous (+/-) for the TGFbeta1 null allele received injections of magnesium silicate (talc), and the extent of abdominal adhesions was determined utilizing a standard grading score. Wild-type (+/+) animals had at least twofold more TGFbeta1 protein in peritoneal fluids at 2 h posttrauma compared to heterozygous (+/-) mice (727 vs. 243 pg TGFbeta1/mg protein by enzyme-linked immunosorbent assay (ELISA) in +/+ and +/- mice, respectively), and had significantly less scar and adhesion formation (p < .05) at 7 days posttrauma (1.8 +/- 0.8 vs. 3.4 +/- 1.4, graded from 0 to 5, in +/+ and +/- mice, respectively). These results demonstrate that haploid insufficiency in TGFbeta1 levels can lead to inappropriate matrix and adhesion production during inflammation, and together with previous studies suggest that any perturbation of normal TGFbeta1 levels can modulate the injury response that regulates the extent of adhesion formation.     

Myostatin (GDF-8) deficiency increases fracture callus size, Sox-5 expression, and callus bone volume

Myostatin (GDF-8) is a negative regulator of skeletal muscle growth and mice lacking myostatin show increased muscle mass. We have previously shown that myostatin deficiency increases bone strength and biomineralization throughout the skeleton, and others have demonstrated that myostatin is expressed during the earliest phase of fracture repair. In order to determine the role of myostatin in fracture callus morphogenesis, we studied fracture healing in mice lacking myostatin. Adult wild-type mice (+/+), mice heterozygous for the myostatin mutation (+/-), and mice homozygous for the disrupted myostatin sequence (-/-) were included for study at two and four weeks following osteotomy of the fibula. Expression of Sox-5 and BMP-2 were significantly upregulated in the fracture callus of myostatin-deficient (-/-) mice compared to wild-type (+/+) mice at two weeks following osteotomy. Fracture callus size was significantly increased in mice lacking myostatin at both two and four weeks following osteotomy, and total osseous tissue area and callus strength in three-point bending were significantly greater in myostatin -/- mice compared to myostatin +/+ mice at four weeks post-osteotomy. Our data suggest that myostatin functions to regulate fracture callus size by inhibiting the recruitment and proliferation of progenitor cells in the fracture blastema. Myostatin deficiency increases blastema size during the early inflammatory phase of fracture repair, ultimately producing an ossified callus having greater bone volume and greater callus strength. While myostatin is most well known for its effects on muscle development, it is also clear that myostatin plays a significant, direct role in bone formation and regeneration.     

Smad3 deficiency attenuates renal fibrosis, inflammation,and apoptosis after unilateral ureteral obstruction

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been implicated in the development of renal fibrosis induced by unilateral ureteral obstruction (UUO). However, there is little information on signaling pathways mediating TGF-beta activity involved in molecular and cellular events leading to renal fibrosis induced by UUO. In this study, we sought to determine whether Smad3, a major signaling component of TGF-beta, mediated renal fibrosis induced by UUO. METHODS: Renal fibrosis, inflammation, and apoptosis induced by UUO were macroscopically and histologically compared between wild-type mice and Smad3 null mice. RESULTS: Gross appearance of the kidney after UUO showed relatively intact kidney in Smad3 null mice [Smad3(-/-) mice] when compared with that of wild-type mice [Smad3(+/+) mice]. Renal interstitial fibrosis based on the interstitial area stained with Aniline-blue or Sirius red solution was significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Deposition of type I and type III collagens were also significantly reduced in the obstructed kidney of Smad3(-/-) mice. In addition, the numbers of myofibroblasts, macrophages, and CD4/CD8 T cells infiltrated into the kidney after UUO were significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Furthermore, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining after UUO showed significantly reduced number of tubular apoptotic cells in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Endogenous Smad pathway was activated in the obstructed kidney after UUO in wild-type mice as judged by the increase of phosphorylated Smad2 or phosphorylated Smad2/3-positive cells in renal interstitial area. CONCLUSION: Smad3 deficiency attenuated renal fibrosis, inflammation, and apoptosis after UUO, suggesting that Smad3 was a key molecule mediating TGF-beta activity leading to real fibrosis after UUO.     

Regulation of inducible class II MHC,costimulatory molecules,and cytokine expression in TGF-beta1 knockout renal epithelial cells: effect of exogenous TGF-beta1

As reports of mice genetically deficient for TGF-beta1 demonstrated aberrant renal class II MHC expression, we investigated inducible class II MHC expression on renal tubular epithelial cells derived from TGF-beta1 knockout (-/-) and wild-type (+/+) mice. IFN-gamma markedly upregulated class II MHC (I-A(b)) expression in both (-/-) and (+/+) tubular epithelial cells. Coincubation studies of (+/+) and (-/-) tubular epithelial cells with IFN-gamma+LPS, or pretreatment of these cells with TGF-beta1, revealed inhibition of IFN-gamma-induced I-A(b) mRNA and cell surface expression that occurred via a decrease in class II transactivator gene expression in both (+/+) and (-/-) tubular epithelial cells. In addition, ICAM-1 was constitutively expressed on both (+/+) and (-/-) tubular epithelial cells and was upregulated by IFN-gamma or IFN-gamma+LPS. ICAM-1 expression in (+/+) and (-/-) tubular epithelial cells, however, was decreased by TGF-beta1. Parallel analysis evaluating B7-1 expression detected low levels of B7-1 in unstimulated (+/+) and (-/-) tubular epithelial cells that were increased by IFN-gamma, LPS, and IFN-gamma+LPS. IFN-gamma+LPS-mediated upregulation of B7-1 was also blocked by pretreatment with TGF-beta1. Cytokine analysis detected significantly higher levels of TNF-alpha and MIP-1alpha mRNA in all treated (-/-) preparations than in (+/+) tubular epithelial cell controls. These studies demonstrate normal patterns of class II MHC, ICAM-1, and B7 expression in TGF-beta1 (-/-) tubular epithelial cells in response to IFN-gamma, LPS, and TGF-beta1. Upregulated cytokine expression at baseline and in response to proinflammatory mediators is apparent in (-/-) tubular epithelial cells, however, and suggests that dysregulation of cytokine expression in inflammatory responses may be a primary event in multifocal inflammation observed in TGF-beta1-deficient animals.     

纳米化仿生诱导骨的构建及促进兔横突间融合研究

目的 探讨纳米化仿生诱导骨促进兔脊柱融合的可行性.方法 成骨化兔脂肪基质细胞(ADSCs)接种在纳米级β磷酸三钙/壳聚糖/聚已内酯基质,构建纳米化仿生诱导骨;建立兔腰5～6横突间融合模型,A组纳米化仿生诱导骨,B组自体髂骨,C组脂肪基质细胞移植,D组空白支架,E组rhBMP-2注射.行影像学、组织学、免疫组化、新骨及诱导因子定量分析等检查.结果 A组融合能力最强,融合率100%、新骨面积比(87.32±1.68)%、BMP含量及力学指标(128±19)、(331.56±3.21)N、(526.78±3.19)Nmm均高于其他组,差异有统计学意义(P＜0.05);B、C组次之,E组新骨生长不明显,融合相对延缓.D组未融合.结论 仿生诱导骨能强化骨再生,促进脊柱融合.     

骨形成蛋白和转化生长因子-β对兔尺骨骨折愈合后生物力学性能的影响

目的 探讨外源性骨形成蛋白(BMP)和转化生长因子—β(TGF—β)对骨折愈合后生物力学性能的影响。方法 36只日本大耳白兔，体重3．5～4．5kg。随机分成4组：空白对照组、TGF—β组、BMP组和TGF—β BMP组，每组9只。采用兔尺骨骨折模型，在骨折局部将聚乳酸(PAL)为载体制备成TGF—β／PAL、BMP／PAL、TGF—β BMP／PLA缓释系统，手术当天将各缓释系统植入骨折区，对照组在相应部位置入PAL，术后50天取材，通过三点弯曲法测骨折愈合后整体骨弯曲结构力学性能的变化。取断端密质骨，用三点弯曲、压缩和拉伸方法测骨试件的材料力学特性的变化，同时测骨痴的几何参数和骨密度的变化来对骨折愈合进行评估。结果 治疗组尺骨的几何参数，整体骨弯曲的破坏载荷、极限强度和弹性棋量，骨试件的弯曲、压缩和拉伸的极限强度，以及弯曲和压缩的弹性棋量明显高于对照组，各治疗组与对照组比较，具有统计学意义(P＜0．01)，TGF—β BMP组高于TGF—β和BMP组；TGF—β组高于BMP组，各治疗组之间两两比较，有统计学意义(P＜0．05)。在骨密度方面，各治疗组与对照组之间无明显差异。结论 兔骨折周围局部应用外源性TGF—β和BMP可促进骨痴形成，增强骨折愈合后骨组织的生物力学强度，TGF—β的作用优于BMP，联合应用优于单用一种生长因子，在促进骨折愈合方面具有协同作用。     

A chemical mutagenesis screen to identify modifier genes that interact with growth hormone and TGF-beta signaling pathways

We describe a phenotype-driven mutagenesis screen in which mice carrying a targeted mutation are bred with ENU-treated males in order to provide a sensitized system for detecting dominant modifier mutations. The presence of initial mutation renders the screening system more responsive to subtle changes in modifier genes that would not be penetrant in an otherwise wild type background. We utilized two mutant mouse models: 1) mice carrying a mutation in growth hormone releasing hormone receptor (Ghrhr) (denoted 'lit' allele, Ghrhr(lit)), which results in GH deficiency; and 2) mice lacking Smad2 gene, a signal transducer for TGF-beta, an important bone growth factor. The Smad2(-/-) mice are lethal and Ghrhr(lit/lit) mice are dwarf, but both Smad2(+/-) and Ghrhr(lit/)(+) mice exhibit normal growth. We injected 6-7 weeks old C57BL/6J male mice with ENU (100 mg/kg dose) and bred them with Ghrhr(lit/)(+) and Smad2(+/-) mice. The F1 mice with Ghrhr(lit/)(+) or Smad2(+/-) genotype were screened for growth and skeletal phenotypes. An outlier was identified as >3 SD units different from wild type control (n=20-30). We screened about 100 F1 mice with Ghrhr(lit/)(+) and Smad2(+/-) genotypes and identified nine outliers. A backcross established heritability of three mutant lines in multiple generations. Among the phenotypic deviants, we have identified a mutant mouse with 30-40% reduced bone size. The magnitude of the bone size phenotype was amplified by the presence of one copy of the disrupted Ghrhr gene as determined by the 2-way ANOVA (p<0.02 for interaction). Thus, a new mouse model has been established to identify a gene that interacts with GH signaling to regulate bone size. In addition, the sensitized screen also demonstrated higher recovery of skeletal phenotypes as compared to that obtained in the classical ENU screen in wild type mice. The discovery of mutants in a selected pathway will provide a valuable tool to not only to discover novel genes involved in a particular process but will also prove useful for the elucidation of the biology of that process.     

Platelet-rich plasma inhibits demineralized bone matrix-induced bone formation in nude mice

BACKGROUND: It is unclear whether platelet-rich plasma is a clinically effective adjunct to osteoinductive agents such as demineralized bone matrix. It contains platelet-derived growth factor (PDGF), which decreases osteoinduction by human demineralized bone matrix in nude-mouse muscle, suggesting that platelet-rich plasma may also have a negative impact. This study tested the hypothesis that platelet-rich plasma reduces demineralized bone matrix-induced bone formation and that this effect varies with donor-dependent differences in platelet-rich plasma and demineralized bone matrix. METHODS: Human platelet-rich plasma was prepared from blood from six men (average age [and standard error of the mean], 29.2 +/- 2.4 years). Platelet numbers were determined, and growth factors were quantified before and after platelet activation. Human demineralized bone matrix from two donors (demineralized bone matrix-1 and demineralized bone matrix-2) was mixed with activated platelet-rich plasma and was implanted bilaterally in the gastrocnemius muscle in eighty male nude mice (eight implants per variable). Fifty-six days after implantation, the hindlimb calf muscles were harvested for histological analysis. Osteoinduction was evaluated with use of a qualitative score and morphometric measurements of ossicle size, new bone formation, and residual demineralized bone matrix. RESULTS: Compared with platelet-poor plasma, platelet-rich plasma preparations exhibited a fourfold increase in the platelet count, a fifteenfold increase in the amount of transforming growth factor-beta, a sixfold increase in the amount of PDGF-BB, a fivefold increase in the amount of PDGF-AA, and a twofold increase in the amount of PDGF-AB. Demineralized bone matrix-1 was more osteoinductive than demineralized bone matrix-2, as determined on the basis of a greater ossicle area. The effect of platelet-rich plasma was either neutral or inhibitory depending on the demineralized bone matrix batch. When used with demineralized bone matrix-1, platelet-rich plasma did not alter the qualitative score or overall ossicle size, but it decreased the new bone area. When used with demineralized bone matrix-2, platelet-rich plasma reduced the qualitative score, ossicle area, and new bone area and increased the amount of residual demineralized bone matrix. The effects on osteoinduction also varied with the donor of the platelet-rich plasma. CONCLUSIONS: Platelet-rich plasma decreased the osteoinductivity of demineralized bone matrix implanted in immunocom-promised mice, and the activities of both demineralized bone matrix and platelet-rich plasma were donor-dependent.     

Compensatory up-regulation of angiotensin II subtype 1 receptors in alpha ENaC knockout heterozygous mice

BACKGROUND: In mice, a partial loss of function of the epithelial sodium channel (ENaC), which regulates sodium excretion in the distal nephron, causes pseudohypoaldosteronism, a salt-wasting syndrome. The purpose of the present experiments was to examine how alpha ENaC knockout heterozygous (+/-) mice, which have only one allele of the gene encoding for the alpha subunit of ENaC, control their blood pressure (BP) and sodium balance. METHODS: BP, urinary electrolyte excretion, plasma renin activity, and urinary adosterone were measured in wild-type (+/+) and heterozygous (+/-) mice on a low, regular, or high sodium diet. In addition, the BP response to angiotensin II (Ang II) and to Ang II receptor blockade, and the number and affinity of Ang II subtype 1 (AT1) receptors in renal tissue were analyzed in both mouse strains on the three diets. RESULTS: In comparison with wild-type mice (+/+), alpha ENaC heterozygous mutant mice (+/-) showed an intact capacity to maintain BP and sodium balance when studied on different sodium diets. However, no change in plasma renin activity was found in response to changes in sodium intake in alpha ENaC +/- mice. On a normal salt diet, heterozygous mice had an increased vascular responsiveness to exogenous Ang II (P < 0.01). Moreover, on a normal and low sodium intake, these mice exhibited an increase in the number of AT1 receptors in renal tissues; their BP lowered markedly during the Ang II receptor blockade (P < 0.01) and there was a clear tendency for an increase in urinary aldosterone excretion. CONCLUSIONS: alpha ENaC heterozygous mice have developed an unusual mechanism of compensation leading to an activation of the renin-angiotensin system, that is, the up-regulation of AT1 receptors. This up-regulation may be due to an increase in aldosterone production.     

The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia-reperfusion injury

Chemokines and their receptors such as CCR2 and CX3CR1 mediate leukocyte adhesion and migration into injured tissue. To further define mechanisms of monocyte trafficking during kidney injury we identified two groups of F4/80-positive cells (F4/80(low) and F4/80(high)) in the normal mouse kidney that phenotypically correspond to macrophages and dendritic cells, respectively. Following ischemia and 3 h of reperfusion, there was a large influx of F4/80(low) inflamed monocytes, but not dendritic cells, into the kidney. These monocytes produced TNF-alpha, IL-6, IL-1alpha and IL-12. Ischemic injury induced in CCR2(-/-) mice or in CCR2(+/+) mice, made chimeric with CCR2(-/-) bone marrow, resulted in lower plasma creatinine levels and their kidneys had fewer infiltrated F4/80(low) macrophages compared to control mice. CX3CR1 expression contributed to monocyte recruitment into inflamed kidneys, as ischemic injury in CX3CR1(-/-) mice was reduced, with fewer F4/80(low) macrophages than controls. Monocytes transferred from CCR2(+/+) or CX3CR1(+/-) mice migrated into reperfused kidneys better than monocytes from either CCR2(-/-) or CX3CR1(-/-) mice. Adoptive transfer of monocytes from CCR2(+/+) mice, but not CCR2(-/-) mice, reversed the protective effect in CCR2(-/-) mice following ischemia-reperfusion. Egress of CD11b(+)Ly6C(high) monocytes from blood into inflamed kidneys was CCR2- and CX3CR1-dependent. Our study shows that inflamed monocyte migration, through CCR2- and CX3CR1-dependent mechanisms, plays a critical role in kidney injury following ischemia reperfusion.     

以纤维蛋白胶为载体复合BMP和bFGF的射型骨修复材料诱导异位成骨的实验研究

目的 :了解以纤维蛋白胶 (Fibrinsealant,FS )为载体的注射型骨修复材料异位诱导成骨的作用 ,为其临床的应用提供实验依据。方法 :实验分组为 :实验组b (FS bFGF bBMP)、对照组b1(FS bBMP)、对照组b2 (bBMP)、实验组r (FS bFGF rhBMP 2 )、对照组r1(FS rhBMP)、对照组r2 (rhBMP)、对照组FS及空白对照组。将各组材料注射或植入小鼠肌袋内 ,采用放射学、形态学、碱性磷酸酶 (ALP)检测等方法对其成骨效应进行研究。结果 :在以bBMP为成骨因子的实验区中 ,实验组b具有高效的骨诱导活性 ,其成骨量显著高于对照组b1、对照组b2、对照组FS及空白对照组(P <0 .0 1) ;在以rhBMP 2为成骨因子的实验区中 ,实验组r同样具有高效的骨诱导活性 ,其成骨量也显著高于对照组r1、对照组r2、对照组FS及空白对照组 (P <0 .0 1)。结论 :以FS为载体复合BMP和bFGF的注射型骨修复材料具有高效的骨诱导活性 ,bFGF可明显增强BMP的骨诱导活性。