The relationship between albuminuria, MCP-1/CCL2, and interstitial macrophages in chronic kidney disease.

Glomerular-derived proteins may activate tubular cells to express the macrophage-directed chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Macrophages at interstitial sites have a central role in directing renal scarring. We have prospectively assessed the relationship between albuminuria, urinary MCP-1/CCL2, interstitial macrophage infiltration, in situ damage, and clinical outcomes in a large group of patients with chronic kidney disease. We studied 215 patients and quantified albumin-creatinine ratio (ACR), urinary MCP-1/CCL2, interstitial macrophage numbers, and in situ damage. ACR correlated with urinary MCP-1/CCL2 (correlation 0.499; P<0.001), interstitial macrophage numbers (correlation 0.481; P<0.001), and index of chronic damage (correlation 0.363; P<0.001). Macrophage numbers closely correlated with in situ damage (correlation 0.755; P<0.001). By multivariate analysis ACR, urinary MCP-1/CCL2, and interstitial macrophage numbers were interdependent. By Kaplan-Meier survival analysis albuminuria, urinary MCP-1/CCL2, interstitial macrophages, and chronic damage predict the outcome. ACR, macrophage numbers, chronic damage, and creatinine independently predicted renal survival. The association of ACR with other variables was strongest in patients with less advanced disease states. There is a close association between albuminuria, urinary MCP-1/CCL2, and interstitial macrophage infiltration with in situ damage and clinical outcomes. These findings support the hypothesis that albuminuria triggers tubular MCP-1/CCL2 expression with subsequent macrophage infiltration. These processes may represent the dominant pathway for the progression of renal injury before the establishment of advanced renal scarring.

The prevalence and clinical significance of autoantibodies to plasminogen activator inhibitor 1 in systemic lupus erythematosus.

We have recently described the novel autoantigen plasminogen activator inhibitor (PAI-1) in systemic lupus erythematosus (SLE). The aim of this study was to determine the prevalence and clinical significance of anti-PAI-1 autoantibodies in patients with SLE. Autoantibodies to recombinant PAI-1 were measured in retrospective sera of 48 lupus patients by immunoassay in order to assess their clinical significance. This showed that 71% of sera from 48 lupus patients had significantly elevated anti-PAI-1 autoantibodies as compared with normal control subjects (P < 0.0001). There was a weak but significant (P < 0.043) correlation with anti-dsDNA autoantibodies. In longitudinal studies, autoantibodies against PAI-1 correlated with clinical parameters measured by the BILAG disease activity index including global clinical score. Our study demonstrates the high frequency of novel autoantibodies to PAI-1 in patients with lupus. The serial clinical correlations with anti-PAI-1 autoantibodies also support the hypothesis that these autoantibodies may play a pathogenic role in lupus.

Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation.

Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus.

Recombinant human transforming growth factor beta 1 modulates bone remodeling in a mineralizing bone organ culture.

TGF-beta 1 increases cell proliferation and collagen synthesis in osteoblast-like cells and bone organ cultures. However, the effects of TGF-beta 1 on bone resorption remain contradictory. Therefore, the exact role that this growth factor plays in the process of bone remodeling is still not clear. We studied the effects of recombinant human TGF-beta 1 (rhTGF-beta 1) on bone formation and resorption in a mineralizing bone organ culture system. Parietal bones from 20-day-old fetal rat calvariae were cultured up to 7 days in serum-free BGJb medium. They responded to a 1 day pulse or continuous treatment of rhTGF-beta 1 with dose-dependent increases in dry weight, [3H]thymidine ([3H]TdR) incorporation, and collagen synthesis. In contrast, rhTGF-beta 1 reduced the calcium content of the bones. This is not due to increased bone resorption but rather to failure of calcium deposition. The following responses occurred at 1 nM rhTGF-beta 1. Dry weight was increased 25-50% after 6 days in culture. DNA synthesis was increased to a maximum at day 1, reaching twofold of the control level. Adding hydroxyurea at day 0 reduced [3H]TdR incorporation in rhTGF-beta 1 treated bones to 20% of the control and indomethacin abrogated the increase in [3H]TdR stimulated by rhTGF-beta 1 to the control level. Both treatments completely blocked the increase in dry weight induced by rhTGF-beta 1 at day 6. rhTGF-beta 1 stimulated collagen synthesis to reach its maximum at day 2, with a twofold increase in [3H]proline incorporation. Basal alkaline phosphatase activity fell continuously in culture, reaching 35% of day 0 level at day 6. Enzyme activity was not altered by rhTGF-beta 1. Morphologic observations by light and electron microscopy confirmed these findings. In summary, rhTGF-beta 1 altered bone remodeling by increasing organic components and decreasing calcification in a mineralizing bone organ culture system.

Human recombinant transforming growth factor-beta 1 modulation of biochemical and cellular events in healing of ulcer wounds.

The effects of recombinant human transforming growth factor beta 1 (rhTGF-beta 1) on wound healing were examined in a rabbit ear ulcer model in which rhTGF-beta 1 was applied to full-thickness biopsy ulcers on the ears. The influence of perichondrium on healing was studied by comparing ulcers with and without perichondrium on 1) formation of total healing wound area (HWA, the newly formed connective and granulation tissues within the ulcer) over time and 2) the amount of collagen synthesized by the wound tissue at day 5. The HWA of ulcers with intact perichondrium increased sharply with time and reached a plateau at day 7, whereas a slower healing occurred in the perichondrium-free model where maximal HWA appeared at day 14. Topical application of 100 ng of rhTGF-beta 1 per wound accelerated healing by increasing HWA in both models. The enhancement of healing by rhTGF-beta 1 was associated with increased collagen synthesis. The percent collagen synthesis in the rhTGF-beta 1 was doubled in the perichondrium-intact ulcers and increased 40% in the perichondrium-free ulcers. DNA synthesis in the perichondrium-intact ulcers was not altered by rhTGF-beta 1 when measured at day 5 by in vitro labeling with [3H]thymidine ([3H]TdR). Autoradiography indicated that the primary cells labeled in the wound tissue were epithelial cells and rhTGF-beta 1 enhanced the migration of these cells from the wound margin towards the center. To evaluate the effects of rhTGF-beta 1 on fibroblasts derived from the granulation tissue of the wound, cells were treated with increasing concentrations of rhTGF-beta 1 and DNA and collagen synthesis were determined. rhTGF-beta 1 elicited a biphasic change in percent collagen synthesis with a maximal increase of 50% at 20 pM followed by a decline. A twofold increase in [3H]TdR incorporation that plateaued at 1 nM was also observed. Our results indicate that the cellular responses to rhTGF-beta 1 differ in vivo and in vitro. The perichondrium-intact ulcers contain more wound tissue and have larger responses to rhTGF-beta 1 stimulation, which allows better examination of biochemical and cellular events. The in vivo mechanisms are multi-factorial, which may involve cell migration and recruitment as results of numerous cell/cell and cell/matrix interactions.

Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: comparison with TGF-beta 1.

The biologic effects of recombinant human bone morphogenetic protein-2b (BMP-2b = BMP-4) were studied and compared with transforming growth factor-beta 1 (TGF-beta 1) in fetal rat osteoblast-like (ROB) cells. Similar to the effects of TGF-beta 1, BMP-2b stimulated DNA and collagen synthesis as well as protein accumulation. Unlike TGF-beta 1, which inhibited alkaline phosphatase activity, BMP-2b enhanced enzyme activity eight-to ninefold over the control level. The present study demonstrates direct actions of BMP-2b on bone-associated cells to stimulate osteogenic phenotypes in vitro and provides a cellular mechanism for the induction of bone formation by BMP-2b in vivo.

Further characterization of insulin-like-growth factor binding proteins in rat osteoblast-like cell cultures: modulation by 17 beta-estradiol and human growth hormone.

Insulin-like growth factors (IGF-I and IGF-II) are endocrine and autocrine factors affecting bone growth and metabolism. Binding proteins for IGFs (IGFBPs) are synthesized by the target tissues of IGF actions. Thus, IGFBPs may act as modulators for the biological functions of IGFs. We have characterized the rat IGFBPs (rIGFBPs) and studied their regulation by 17 beta-estradiol (beta E2) and human GH (hGH) in rat osteoblast-like (ROB) cell cultures. ROB cells were prepared from 19- to 20-day fetal rat calvariae by sequential collagenase digestion and studies were performed on the serum and phenol red-free conditioned medium of confluent cultures. [125I]IGF-I ligand blots showed that the major rIGFBP in the ROB is a nonglycosylated protein of 31 kilodaltons. This protein was immunoprecipitated by a specific antibody to rIGFBP-2 and messenger RNA for rIGFBP-2 was detected by RNA hybridization indicating that the rIGFBP-2 is the major rIGFBP of ROB. A minor band at 24 kilodaltons is likely to be the rat homologue of the newly isolated inhibitory IGFBP-4. The predominant glycosylated adult form of rIGFBPs of rat serum, rIGFBP-3, was undetectable. When cultures were treated with beta E2 for 2 days, there was a dose-dependent biphasic response which showed an inhibition of the rIGFBP-2 at low doses of 10(-11) to 10(-9) M and a stimulation at 10(-6) M. These changes in rIGFBP-2 parallel the changes in the endogenous IGF-I level. rIGFBP-2 level was not affected by 17 alpha-estradiol at the same concentration range. hGH, on the other hand stimulated the levels of rIGFBP-2 and rIGFBP-4 at doses ranging from 10(-11) to 10(-9) M without changing the IGF-I secretion. The alteration of the rIGFBPs by beta E2 and hGH suggests a role for these hormones in bone by modulating the biological functions of IGFs via their binding proteins.

Dexamethasone and 1,25-dihydroxyvitamin D3 modulation of insulin-like growth factor-binding proteins in rat osteoblast-like cell cultures.

Using the method of Western ligand blot, we have found that the major form of insulin-like growth factor-binding protein (IGF-BP) secreted by rat osteoblastic-like cells in culture is a 31-kDa protein that is immunologically identical to BP-2, the binding protein originally identified in conditioned medium of Buffalo rat liver cells (BRL-3A). Two minor forms of IGF-BPs with apparent mol wt of 24 kDa (BP-24) and 42 kDa (BP-42) have also been identified. The amount of IGF-BPs in serum-free conditioned medium increased 3-fold on day 3 compared to the day 1 level. We also studied the modulation of IGF-BPs by dexamethasone (DEX), 1,25-dihydroxyvitamin D [1,25-(OH)2D3], and insulin-like growth factor-I (IGF-I). DEX coordinately reduced the level of IGF-BPs in a dose- and time-dependent manner, which resulted in less than 10% of the BP-2 remaining at 100 nM. In contrast, 1,25-(OH)2D3 at 100 nM enhanced the amount of BP-2 by 1-fold. In combined treatments, 1,25-(OH)2D3 at 10 nM was unable to antagonize the inhibitory effect of DEX in the dose range of 1-10 nM. IGF-I, at 1 and 10 nM, proved to be a potent stimulator of all IGF-BPs, and at 10 nM, it completely reversed the inhibition by 100 nM DEX. Although the roles of IGF-BPs have not been clearly defined in bone cells, they are capable of modulating the biological actions of IGFs in other cell culture systems. Modulation of the IGF-BP level by DEX, 1,25-(OH)2D3, and IGF-I suggests important roles for these binding proteins in altering IGF-I action in rat osteoblast-like cell cultures.

Accelerated healing of ulcer wounds in the rabbit ear by recombinant human transforming growth factor-beta 1.

A dermal ulcer wound-healing model was established in rabbit ear to examine the effects of recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) in wound healing. Histomorphometric examination of the wounds indicate a biphasic healing response 7 days after a single application of rhTGF-beta 1 at the time of wounding. Statistically significant healing occurred at 5-100 ng but not at higher doses of 500 or 1000 ng rhTGF-beta 1/wound. Enhanced collagen synthesis as determined by [3H]proline incorporation occurred at 15 and 25 ng and was significantly depressed at 500 ng rhTGF-beta 1/wound. Multiple doses of 100 ng rhTGF-beta 1 applied to the wound at the time of wounding and for 3 days after wounding provided results comparable to the single application of growth factor. Delaying treatment 24 hr after wounding did not enhance wound healing compared with vehicle. Our findings suggest that rhTGF-beta 1 can be a valuable growth factor to improve the healing of ulcer wounds.

An unusual manifestation of congenital abdominal aortic coarctation.

Coarctation of the abdominal aorta is uncommon, occurring in only 0.5-2% of aortic coarctations. This disease is usually manifest by upper body hypertension. A case report of a thirty-three-year-old female who had the unusual manifestation of peripheral emboli to both lower extremities secondary to fibrin emboli from her infrarenal segmental aortic coarctation was presented. This is the only case we know of to be manifest in this manner. No others could be found in an English-language literature search.