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Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear. In this study, we used a renal bilateral ischemia-reperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b(+) cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies. Macrophage populations were isolated from kidney homogenates by fluorescence-activated cell sorting for whole genome microarray analysis. The CD11b(+)/Ly6C(high) population associated with the onset of renal injury and increase in proinflammatory cytokines, whereas the CD11b(+)/Ly6C(intermediate) population peaked during kidney repair. The CD11b(+)/Ly6C(low) population emerged with developing renal fibrosis. Principal component and hierarchical cluster analyses identified gene signatures unique to each population. The CD11b(+)/Ly6C(intermediate) population had a distinct phenotype of wound healing, confirmed by results of studies inhibiting the macrophage colony-stimulating factor 1 receptor,whereas the CD11b(+)/Ly6C(low) population had a profibrotic phenotype. All populations, including the CD11b(+)/Ly6C(high) population, carried differential inflammatory signatures. The expression of M2-specific markers was detected in both the CD11b(+)/Ly6C(intermediate) and CD11b(+)/Ly6C(low) populations, suggesting these in vivo populations do not fit into the traditional classifications defined by in vitro systems. Results of this study in a renal ischemia-reperfusion injury model allow phenotype and function to be assigned to CD11b(+)/Ly6C(+) monocyte/macrophage populations in the pathophysiology of disease after AKI.

Divergent effects of low versus high dose anti-TGF-β antibody in puromycin aminonucleoside nephropathy in rats.BackgroundTransforming growth factor-β (TGF-β) modulates immune/inflammatory cells, promotes extracellular matrix (ECM) accumulation, and is increased in fibrotic organs. Here we report the effects of administering a puromycin aminonucleoside nephropathy (PAN)-specific TGF-β neutralizing antibody on glomerulosclerosis in vivo.MethodsAdult male Sprague-Dawley rats underwent uninephrectomy (Nx) followed by intraperitoneal PAN at weeks 2, 6, 7 and 8. Rats were treated with either high (5mg/kg body weight) (N = 9) or low (0.5mg/kg body weight) (N = 7) dose TGF-β antibody intraperitoneally three times weekly until sacrifice at week 10. A PAN untreated control group (N = 7) was dosed with an isotype specific, null antibody. The nephrectomy samples were studied as normal kidney control (NL) (N = 5). Rats undergoing left kidney Nx (N = 5) only were also included as age-matched control. Renal function and morphology were assessed, and molecular studies performed.ResultsSystolic blood pressure was increased in parallel over time in all groups (at 10weeks, control 137 ± 10mm Hg; high 129 ± 4mm Hg; low 137 ± 3mm Hg) (P = NS). Both TGF-β antibody treatments decreased renal cortex mRNA expressions similarly for TGF-β1, TGF-β2, and collagen III (TGF-β1, control 0.36 ± 0.02mm Hg; high 0.19 ± 0.01mm Hg; low 0.19 ± 0.02mm Hg; P < 0.01 low and high vs. control; TGF-β2, control 0.38 ± 0.03mm Hg; high 0.19 ± 0.02mm Hg; low 0.20 ± 0.03mm Hg; P < 0.01 low and high vs. control; and collagen III, control 0.33 ± 0.01mm Hg; high 0.14 ± 0.01mm Hg; low 0.19 ± 0.01mm Hg; P < 0.01 low and high vs. control; P < 0.05 low vs. high, data expressed as mRNA normalized density units vs. 18S RNA). However, only low dose TGF-β antibody improved renal function and sclerosis measured by serum creatinine and creatinine clearance (serum creatinine, control 2.3 ± 0.5mg/dL; high 2.5 ± 0.5mg/dL; low 0.8 ± 0.1mg/dL; P < 0.05 low vs. control and high; creatinine clearance, control 0.44 ± 0.11mL/min; high 0.70 ± 0.26mL/min; low 1.34 ± 0.30mL/min; P < 0.05 low vs. control, P = NS vs. high). In parallel, sclerosis index (0 to 4+ scale) was improved in low dose (control 2.67 ± 0.27; high 2.37 ± 0.30; low 1.78 ± 0.24; P < 0.05 low vs. control). This improved function and structure was linked to decreased glomerular infiltrating macrophages (0 to 4+ score, control 2.3 ± 0.2; high 1.8 ± 0.4; low 0.8 ± 0.1; P < 0.01 low vs. control; P < 0.05 low vs. high; P = NS high vs. control). Further, plasminogen activator inhibitor-1 (PAI-1) mRNA expression in renal cortex was attenuated after low dose TGF-β antibody treatment compared to control and high dose group (PAI-1/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratio, NL 0.18 ± 0.003; control 0.45 ± 0.03; high 0.40 ± 0.04; low 0.23 ± 0.01; P < 0.05 low vs. control and high). Matrix metalloproteinase-9 (MMP-9) activity was maintained at higher levels in kidneys of the low dose TGF-β antibody-treated group.ConclusionThese results show an in vivo dose-response with an agent that blocks the biologic activity of TGF-β. Higher dose of TGF-β antibody was without beneficial effect, suggesting that TGF-β–mediated effects on PAI-1 and macrophage influx are bimodal and closely regulated. Given that both antibody doses reduced the expression of TGF-β isoforms and collagen III production, but only low dose ameliorated histologic sclerosis, it appears that pharmacologic effects of anti-TGF-β antibody on matrix synthesis and degradation are not equivalent.

Mice genetically deficient in TGF-beta1 or TGF-beta signaling capacity in T or B cells demonstrate profound immune dysregulation, as evidenced by increased lymph node size, expression of markers of memory/activation on T cells, inflammation in a variety of tissues and development of autoantibodies. However, this constant and complete lack of TGF-beta1 or TGF-betaR signaling may not reflect effects of TGF-beta neutralization using antibodies in mature animals. Thus, the present studies were designed to determine if administration of an anti-TGF-beta monoclonal antibody (neutralizes TGF-beta1, 2 and 3) to mature, normal mice results in evidence of immune dysregulation or immune-mediated pathology. An initial study examined daily administration of 0.25, 0.75 and 2.5 mg/kg of anti-TGF-beta to mice for three weeks, achieving blood levels of as high as 9 mg/ml. Comprehensive hematological and histopathological evaluation showed no evidence of pathology. A second study was designed to extend the antibody treatment period and further examine the functional status of the immune system. Mice were injected with 1 mg/mouse (approximately 50 mg/kg) of anti-TGF-beta (1D11) three times per week achieving circulating blood levels of 1-2 mg/ml. Many parameters of immune status were assessed, including natural killer (NK) cell activity, lymphocyte proliferative responses, phagocytic activity, phenotypic assessment of leukocyte subsets, and serum measurements of proinflammatory cytokines, autoantibodies and immunoglobulin isotypes. In addition, histopathological assessment of heart, lungs, liver, kidney, salivary glands, skin, spleen and lymph nodes was also performed. Very few of the multiple immune parameters examined showed detectable changes in anti-TGF-beta-treated mice. Changes that were observed were primarily restricted to the spleen and included increased spleen cell recoveries, increased percentages of macrophages, decreased percentages of NK cells, decreased phagocytic activity, decreased proliferative responses to mitogens and slight increases in T and B cells displaying an activated phenotype. Many of these same parameters examined in the lymph nodes were not altered by the anti-TGF-beta treatment. The thymus was decreased in size, but altered only slightly in one population of developing T cells. Most of the changes observed were modest and returned to control levels after discontinuation of treatments. The only serological finding was an increase in IgA levels in anti-TGF-beta-treated mice, but not in any other isotype. Finally, there was no evidence of increased inflammation in any of the peripheral tissues examined in the anti-TGF-beta-treated mice. In conclusion, although there were changes in some of the immunological parameters examined in these studies, they were few and typically reversed following discontinuation of treatment. The modest nature of the changes observed in these studies is particularly evident when compared to published data of those same parameters examined in mice genetically deficient in TGF-beta1 or mice having TGF-beta unresponsive T or B cells. Thus, there does not appear to be any significant immune dysregulation detectable after long-term antibody-mediated neutralization of TGF-beta in normal mice.

Antibody to transforming growth factor-β ameliorates tubular apoptosis in unilateral ureteral obstruction. Background Unilateral ureteral obstruction (UUO) is characterized by progressive renal atrophy, renal interstitial fibrosis, an increase in renal transforming growth factor-β (TGF-β), and renal tubular apoptosis. The present study was undertaken to determine the effect of a monoclonal antibody to TGF-β (1D11) in UUO. Methods Mechanical stretch was applied to tubular epithelial cells (NRK-52E) by a computer-assisted system. Three doses of 1D11 (either 0.5, 2, or 4 mg/rat) were administered to rats one day prior to UUO and every two days thereafter, and kidneys were harvested at day 13. Fibrosis was assessed by measuring tissue hydroxyproline and mRNA for collagen and fibronectin. Apoptosis was assessed with the terminal deoxy transferase uridine triphosphate nick end-labeling assay. TGF-β levels were determined by bioassay. Western blot and immunostaining were used to identify proliferating cell nuclear antigen (PCNA), p53, bcl-2, and inducible nitric oxide synthase (iNOS). Results Stretch significantly induced apoptosis in NRK-52E cells, which was accompanied by an increased release of TGF-β; 1D11 (10 μg/mL) totally inhibited stretch-induced apoptosis. Control obstructed kidney contained 20-fold higher TGF-β as compared with its unobstructed kidney; 1D11 neutralized tissue TGF-β of the obstructed kidney. Control obstructed kidney exhibited significantly more fibrosis and tubular apoptosis than its unobstructed counterpart, which was blunted by 1D11. In contrast, 1D11 significantly increased tubular proliferation. p53 immunostaining was localized to renal tubular nuclei of control obstructed kidney and was diminished by 1D11. In contrast, bcl-2 was up-regulated in the 1D11-treated obstructed kidney. Total NOS activity and iNOS activity of the obstructed kidney were increased by 1D11 treatment. Conclusion The present study strongly suggests that an antibody to TGF-β is a promising agent to prevent renal tubular fibrosis and apoptosis in UUO.

A group of specialized mesenchymal cells located at the root of the mammalian hair follicle, known as the follicular or dermal papillary cells, are involved in regulating the hair cycle, during which keratinocytes of the lower follicle undergo proliferation, degeneration and regrowth. Using the arbitrarily primed-PCR approach, we have identified a 1.3 kb messenger RNA that is present in large quantities in cultured rat follicular papillary cells, but not in skin fibroblasts. This mRNA encodes nexin 1, a potent protease inhibitor that can inactivate several growth-modulating serine proteases including thrombin, urokinase and tissue plasminogen activator. In situ hybridization showed that nexin 1 message is accumulated in the follicular papilla cells of anagen follicles, but is undetectable in keratinocytes or other skin mesenchymal cells. In addition, nexin 1 message level varies widely among several immortalized rat vibrissa papillary cell lines, and these levels correlate well with the reported abilities of these cell lines to support in vivo follicular reconstitution. These results suggest a possible role of nexin 1 in regulating hair follicular growth.

Fibrosis is a final common pathway leading to loss of kidney function, in which the fibrogenic cytokine, transforming growth factor β (TGF-β), plays a central role. While previous studies showed that TGF-β antagonism by various means prevents fibrosis in mouse models, clinical approaches based on these findings remain elusive. 1D11 is a neutralizing antibody to all three isoforms of TGF-β. In both adriamycin (ADR)-induced nephropathy and NEP25 podocyte ablation nephropathy, thrice-weekly intraperitoneal administration of 1D11 from the day of disease induction until the mice were sacrificed (day 14 for ADR and day 28 for NEP25), significantly reduced glomerular COL1A2 mRNA accumulation and histological changes. Consistent with our previous findings, proteinuria remained overt in the mice treated with 1D11, suggesting distinct mechanisms for proteinuria and fibrogenesis. Podocyte numbers determined by WT1 staining were significantly reduced in NEP25-model glomeruli as expected, while WT1-positive cells were preserved in mice receiving 1D11. Even when 1D11 was administered after the onset of proteinuria on day 3, 1D11 preserved WT1-positive cell numbers in glomeruli and significantly reduced glomerular scar score (2.5 ± 0.2 [control IgG] vs. 1.8 ± 0.2 [1D11], P < 0.05) and glomerular COL1A2 mRNA expression (19.3 ± 4.4 [control IgG] vs. 8.4 ± 2.4 [1D11] fold increase over the healthy control, P < 0.05). Transmission electron microscopy revealed loss of podocytes and denuded glomerular basement membrane in NEP25 mice with disease, whereas podocytes remained attached to the basement membrane, though effaced and swollen, in those receiving 1D11 from day 3. Together, these data suggest that TGF-β neutralization by 1D11 prevents glomerular fibrosis even when started after the onset of proteinuria. While overt proteinuria and podocyte effacement persist, 1D11 prevents total podocytes detachment, which might be a key event activating fibrogenic events in glomeruli.

The nude mouse graft model for testing the hair-forming ability of selected cell populations has considerable potential for providing insights into factors that are important for hair follicle development and proper hair formation. We have developed a minimal component system consisting of immature hair follicle buds from newborn pigmented C57BL/6 mice and adenovirus E1A-immortalized rat vibrissa dermal papilla cells. Hair follicle buds contribute to formation of hairless skin when grafted alone or with Swiss 3T3 cells, but produce densely haired skin when grafted with a fresh dermal cell preparation. The fresh dermal cell preparation represents the single cell fraction after hair follicles have been removed from a collagenase digest of newborn mouse dermis. It provides dermal papilla cells, fibroblasts, and possibly other important growth factor-producing cell types. Rat vibrissa dermal papilla cells supported dense hair growth at early passage in culture but progressively lost this potential during repeated passage in culture. Of 19 E1A-immortalized, clonally derived rat vibrissa dermal papilla cell lines, the four most positive clones supported hair growth to the extent of approximately 200 to 300 hairs per 1–2 cm 2 graft area. The remaining clones were moderately positive (five clones), weakly positive (three clones), or negative (seven clones). Swiss 3T3 cells prevented contraction of the graft area but did not appear to affect the number of hairs in the graft site produced by dermal papilla cells plus hair follicle buds alone. The relatively low hair density (estimated 1–5% of normal) resulting from grafts of hair follicle buds with the most positive of the immortalized dermal papilla cell clones compared to fresh dermal cells suggests that optimal reconstitution of hair growth requires some function of dermal papilla cells partially lost during the immortalization process and possibly the contribution of other cell types present in the fresh dermal cell preparation, which is not supplied by the Swiss 3T3 cells. The current graft system, comprising hair follicle buds and immortalized dermal papilla cell clones, provides an assay for positive or negative influences on hair growth exerted by added selected cell types, growth factors, or other substances. Characterization of the phenotype of the dermal papilla cell lines, which differ in their ability to support hair growth when grafted with hair follicle buds, may provide insight into specific dermal papilla cell properties important for their function in this system.

Combinations of cultured and uncultured epidermal and dermal cell preparations from newborn and perinatal mice were grafted onto the backs of athymic nude mouse hosts to elucidate the cellular requirements for skin appendage formation. All epidermal populations studied, including a total epidermal keratinocyte preparation from trypsin-split skin, developing hair follicle buds isolated from epidermis, and preformed hair follicles isolated from dermis, make haired skin when grafted with fresh dermal cells. Only pre-formed hair follicles produce haired skin on grafts without an additional dermal component. Hair follicle buds grafted alone or with cultured dermal cells will reconstitute skin but without appendage formation. Thus, cells or factors present in fresh, but not cultured, dermal cells are essential for supporting hair growth from budding follicles, whereas more developed (pre-formed) follicles appear to contain all the necessary components for hair formation. Dissociation of isolated hair follicles by trypsin/ethylenediaminetetraacetic acid prior to grafting is permissive for hair growth, suggesting that follicle cells can be re-induced or reassociate in vivo. Dermal papilla cells, microdissected from rat vibrissal follicles and cultured for up to 14 passages, stimulate hair growth from follicle buds and influence the quality of hair growth from pre-formed hair follicles. Thus, dermal papilla cells maintain inductive capacity in culture and contribute to the reconstituted skin. This reconstitution model should be useful for identifying cell populations within the hair follicle compartment necessary for hair growth and for examining the effects of specific gene products on hair follicle growth and development in vivo.

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor; its overexpression has been implicated in many diseases, making it a desirable target for therapeutic neutralization. In initial safety studies, mice were chronically treated (three times per week) with high doses (50 mg/kg) of a murine, pan-neutralizing, anti-TGF-beta antibody. Nine weeks after the initiation of treatment, a subset of mice exhibited weight loss that was concurrent with decreased food intake. Histopathology revealed a unique, nonneoplastic cystic epithelial hyperplasia and tongue inflammation, as well as dental dysplasia and epithelial hyperplasia and inflammation of both the gingiva and esophagus. In an effort to determine the cause of this site-specific pathology, we examined TGF-beta expression in these tissues and saliva under normal conditions. By immunostaining, we found higher expression levels of active TGF-beta1 and TGF-beta3 in normal tongue and esophageal submucosa compared with gut mucosal tissues, as well as detectable TGF-beta1 in normal saliva by Western blot analysis. Interestingly, mast cells within the tongue, esophagus, and skin co-localized predominantly with the TGF-beta1 expressed in these tissues. Our findings demonstrate a novel and restricted pathology in oral and esophageal tissues of mice chronically treated with anti-TGF-beta that is associated with basal TGF-beta expression in saliva and by mast cells within these tissues. These studies illustrate a previously unappreciated biological role of TGF-beta in maintaining homeostasis within both oral and esophageal tissues.

Background: In diabetic rats with maximal activation of RAS induced by uninephrectomy, late treatment with anti-TGFβ antibody limited renal injury only when combined with ACE inhibitor. We investigated whether in a two-kidney diabetic model the time at which treatment started predicted the response to TGFβ antagonist. Methods: 27 weeks after streptozotocin injection, animals had mild proteinuria and were randomized to receive irrelevant antibody, anti-TGFβ antibody (1D11) or enalapril till 52 weeks (early treatment). The effect of agents alone or combined was also evaluated at the time of overt proteinuria (late treatment, 52–61 weeks). Results: When given early, 1D11 displayed marked antihypertensive and antiproteinuric effects. Glomerulosclerosis was reduced to the extent that a remarkable percentage of glomeruli without sclerosis appeared after treatment. Podocyte number was normalized. Renoprotection of 1D11 was comparable to enalapril. Despite control of blood pressure, in late treatment single agents did not reduce proteinuria significantly. Glomerulosclerosis and podocyte loss were partially limited by 1D11 or enalapril, but full protection was achieved by combination. Conclusions: Renoprotective effect of TGFβ antagonism crucially depends on the time at which treatment started. Effectiveness of early treatment with 1D11 would indicate that TGFβ is a major mediator of damage in early diabetes. To tackle the renal damage in the phase of advanced disease, a combined treatment with ACE inhibitor is needed.