Guoping Zheng

Disruption of E-Cadherin by Matrix Metalloproteinase Directly Mediates Epithelial-Mesenchymal Transition Downstream of Transforming Growth Factor-β1 in Renal Tubular Epithelial Cells

Epithelial-mesenchymal transition (EMT) plays an important role in organ fibrosis, including that of the kidney. Loss of E-cadherin expression is a hallmark of EMT; however, whether the loss of E-cadherin is a consequence or a cause of EMT remains unknown, especially in the renal system. In this study, we show that transforming growth factor (TGF)-beta1-induced EMT in renal tubular epithelial cells is dependent on proteolysis. Matrix metalloproteinase-mediated E-cadherin disruption led directly to tubular epithelial cell EMT via Slug. TGF-beta1 induced the proteolytic shedding of E-cadherin, which caused the nuclear translocation of beta-catenin, the transcriptional induction of Slug, and the repression of E-cadherin transcription in tubular epithelial cells. These findings reveal a direct role for E-cadherin and for matrix metalloproteinases in causing EMT downstream of TGF-beta1 in fibrotic disease. Specific inhibition rather than activation of matrix metalloproteinases may offer a novel approach for treatment of fibrotic disease.

E-Cadherin/β-Catenin Complex and the Epithelial Barrier

E-Cadherin/β-catenin complex plays an important role in maintaining epithelial integrity and disrupting this complex affect not only the adhesive repertoire of a cell, but also the Wnt-signaling pathway. Aberrant expression of the complex is associated with a wide variety of human malignancies and disorders of fibrosis resulting from epithelial-mesenchymal transition. These associations provide insights into the complexity that is likely responsible for the fibrosis/tumor suppressive action of E-cadherin/β-catenin.

IL-10/TGF-beta-modified macrophages induce regulatory T cells and protect against adriamycin nephrosis.

IL-10/TGF-beta-modified macrophages, a subset of activated macrophages, produce anti-inflammatory cytokines, suggesting that they may protect against inflammation-mediated injury. Here, macrophages modified ex vivo by IL-10/TGF-beta (IL-10/TGF-beta Mu2) significantly attenuated renal inflammation, structural injury, and functional decline in murine adriamycin nephrosis (AN). These cells deactivated effector macrophages and inhibited CD4+ T cell proliferation. IL-10/TGF-beta Mu2 expressed high levels of the regulatory co-stimulatory molecule B7-H4, induced regulatory T cells from CD4+CD25- T cells in vitro, and increased the number of regulatory T cells in lymph nodes draining the kidneys in AN. The phenotype of IL-10/TGF-beta Mu2 did not switch to that of effector macrophages in the inflamed kidney, and these cells did not promote fibrosis. Taken together, these data demonstrate that IL-10/TGF-beta-modified macrophages effectively protect against renal injury in AN and may become part of a therapeutic strategy for chronic inflammatory disease.

CD4+CD25+ Regulatory T Cells Protect against Injury in an Innate Murine Model of Chronic Kidney Disease

Studies of mechanisms of disease regulation by CD4+CD25+ regulatory T cells (Treg) have been focused on their interaction with effector T cells; however, the possibility that regulation might involve noncognate cells has not been explored in detail. This study investigated the effect of CD4+CD25+ Treg on macrophage proinflammatory properties and phenotype in vitro and found that they modulate macrophages by inhibiting their activation, leading to reduced proinflammatory cytokine production and a downregulated effector phenotype. For testing the in vivo significance of this effect, CD4+CD25+ T cells that expressed high levels of Foxp3 were reconstituted into SCID mice after induction of Adriamycin nephropathy, a noncognate model of chronic renal disease. CD4+CD25+ T cells significantly reduced glomerular and interstitial injury. In addition, there was a significant fall in the number of macrophages in both the glomeruli and interstitium of SCID mice that were reconstituted with Treg as compared with the Adriamycin alone group. Blockade of TGF-beta using neutralizing antibodies significantly impaired the protective effect of Treg. These findings delineate a TGF-beta-dependent Treg-macrophage inhibitory interaction that can explain cognate-independent protection by Treg.

Macrophage matrix metalloproteinase-9 mediates epithelial-mesenchymal transition in vitro in murine renal tubular cells.

As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial-mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-beta. Transforming growth factor-beta-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis.

Characterization of murine macrophages from bone marrow, spleen and peritoneum

BackgroundMacrophages have heterogeneous phenotypes and complex functions within both innate and adaptive immune responses. To date, most experimental studies have been performed on macrophages derived from bone marrow, spleen and peritoneum. However, differences among macrophages from these particular sources remain unclear. In this study, the features of murine macrophages from bone marrow, spleen and peritoneum were compared.ResultsWe found that peritoneal macrophages (PMs) appear to be more mature than bone marrow derived macrophages (BMs) and splenic macrophages (SPMs) based on their morphology and surface molecular characteristics. BMs showed the strongest capacity for both proliferation and phagocytosis among the three populations of macrophage. Under resting conditions, SPMs maintained high levels of pro-inflammatory cytokines expression (IL-6, IL-12 and TNF-α), whereas BMs produced high levels of suppressive cytokines (IL-10 and TGF-β). However, SPMs activated with LPS not only maintained higher levels of (IL-6, IL-12 and TNF-α) than BMs or PMs, but also maintained higher levels of IL-10 and TGF-β.ConclusionsOur results show that BMs, SPMs and PMs are distinct populations with different biological functions, providing clues to guide their further experimental or therapeutic use.

Discrete functions of M2a and M2c macrophage subsets determine their relative efficacy in treating chronic kidney disease

Two types of alternatively activated macrophages, M(2a) induced by IL-4/IL-13 and M(2c) by IL-10/TGF-β, exhibit anti-inflammatory functions in vitro and protect against renal injury in vivo. Since their relative therapeutic efficacy is unclear, we compared the effects of these two macrophage subsets in murine adriamycin nephrosis. Both subsets significantly reduced renal inflammation and renal injury; however, M(2c) macrophages more effectively reduced glomerulosclerosis, tubular atrophy, interstitial expansion, and proteinuria than M(2a) macrophages. The M(2c) macrophages were also more effective than M(2a) in reduction of macrophage and CD4(+) T-cell infiltration in kidney. Moreover, nephrotic mice treated with M(2c) had a greater reduction in renal fibrosis than those treated with M(2a). M(2c) but not M(2a) macrophages induced regulatory T cells (Tregs) from CD4(+)CD25(-) T cells in vitro, and increased Treg numbers in local draining lymph nodes of nephrotic mice. To determine whether the greater protection with M(2c) was due to their capability to induce Tregs, the Tregs were depleted by PC61 antibody in nephrotic mice treated with M(2a) or M(2c). Treg depletion diminished the superior effects of M(2c) compared to M(2a) in protection against renal injury, inflammatory infiltrates, and renal fibrosis. Thus, M(2c) are more potent than M(2a) macrophages in protecting against renal injury due to their ability to induce Tregs.

By Homing to the Kidney, Activated Macrophages Potently Exacerbate Renal Injury

Macrophages are important mediators of injury in most types of human kidney diseases; however, the pathogenic importance of both macrophage number and activation status is unknown. To examine this question, severe-combined immunodeficient mice with adriamycin nephrosis, an experimental model of human focal segmental glomerulosclerosis, were treated intravenously with either resting (1 x 10(6) to 5 x 10(6)) or activated (1 x 10(3) to 1 x 10(6)) macrophages on day 6 postadriamycin administration, and the effects on kidney injury were examined. On day 28, renal injury was worse in the group that received activated macrophages at doses as low as 1 x 10(4) macrophages per mouse compared with control adriamycin nephrotic mice. However, treatment with resting macrophages at doses as high as 5 x 10(6) macrophages per mouse had no significant effect on either renal histology or function. The transferred activated macrophages homed to inflamed kidneys during the middle-to-late stages of the disease, but such homing was not observed for resting macrophages. This study of in vivo cell adoptive transfer supports the importance of macrophage activation status over macrophage number in causing renal injury. These data suggest that therapeutic strategies for treating progressive kidney diseases should target activated macrophages.

Matrix metalloproteinase-9 of tubular and macrophage origin contributes to the pathogenesis of renal fibrosis via macrophage recruitment through osteopontin cleavage.

A pro-fibrotic role of matrix metalloproteinase-9 (MMP-9) in tubular cell epithelial–mesenchymal transition (EMT) is well established in renal fibrosis; however studies from our group and others have demonstrated some previously unrecognized complexity of MMP-9 that has been overlooked in renal fibrosis. Therefore, the aim of this study was to determine the expression pattern, origin and the exact mechanism underlying the contribution of MMP-9 to unilateral ureteral obstruction (UUO), a well-established model of renal fibrosis via MMP-9 inhibition. Renal MMP-9 expression in BALB/c mice with UUO was examined on day 1, 3, 5, 7, 9, 11 and 14. To inhibit MMP-9 activity, MMP-2/9 inhibitor or MMP-9-neutralizing antibody was administered daily for 4 consecutive days from day 0–3, 6–9 or 10–13 and tissues harvested at day 14. In UUO, there was a bi-phasic early- and late-stage upregulation of MMP-9 activity. Interestingly, tubular epithelial cells (TECs) were the predominant source of MMP-9 during early stage, whereas TECs, macrophages and myofibroblasts produced MMP-9 during late-stage UUO. Early- and late-stage inhibition of MMP-9 in UUO mice significantly reduced tubular cell EMT and renal fibrosis. Moreover, MMP-9 inhibition caused a significant reduction in MMP-9-cleaved osteopontin and macrophage infiltration in UUO kidney. Our in vitro study showed MMP-9-cleaved osteopontin enhanced macrophage transwell migration and MMP-9 of both primary TEC and macrophage induced tubular cell EMT. In summary, our result suggests that MMP-9 of both TEC and macrophage origin may directly or indirectly contribute to the pathogenesis of renal fibrosis via osteopontin cleavage, which, in turn further recruit macrophage and induce tubular cell EMT. Our study also highlights the time dependency of its expression and the potential of stage-specific inhibition strategy against renal fibrosis.

Transfused macrophages ameliorate pancreatic and renal injury in murine diabetes mellitus.

Background: Alternatively activated macrophages (M2 macrophages) are able to reduce renal injury in murine adriamycin nephropathy. However, the effect of M2 macrophages in other renal diseases such as diabetic nephropathy remains unknown. Methods: Macrophages were separated from splenocytes and polarized with IL-4 and IL-13 into a protective phenotype. Mice underwent adoptive transfer with M2 macrophages, and then diabetes was induced by tail vein injection with streptozotocin (STZ). Blood glucose levels were monitored daily. Mice were sacrificed at week 10 after STZ. Renal function and histopathological injury were assessed quantitatively. Results: Transfused M2 macrophages accumulated progressively in kidneys for up to 10 weeks after STZ. Kidneys from diabetic mice transfused with M2 macrophages had less tubular atrophy, glomerular hypertrophy and interstitial expansion than did control diabetic mice. M2 macrophages suppressed the development of interstitial fibrosis. In addition, the degree of pancreatic islet injury, as assessed by insulin staining, haemoglobin A1c and blood glucose was reduced after transfusion of M2 macrophages. In vivo, activation of kidney endogenous macrophage cytokine expression was inhibited by M2 macrophages. Conclusion: Our findings show that M2 macrophages can protect against islet and renal injury in streptozotocin-induced diabetes, providing a potential therapeutic strategy for diabetes and diabetic nephropathy.