Vincent W.S. Lee

Target of rapamycin inhibitors (sirolimus and everolimus) for primary immunosuppression of kidney transplant recipients: a systematic review and meta-analysis of randomized trials.

Background. Target of rapamycin inhibitors (TOR-I) have a novel mode of action but uncertain clinical role. We performed a systematic review of randomized trials where immunosuppressive regimens containing TOR-I were compared with other regimens as initial therapy for kidney transplant recipients. Methods. Databases (inception, June 2005) and conference proceedings (1996–2005) were searched. Two independent reviewers assessed trials for eligibility and quality. Results at 1 year, are expressed as relative risk (RR), where values <1 favor TOR-I, or lower dose of TOR-I, and for continuous outcomes are expressed as weighted mean difference (WMD), both expressed with 95% confidence intervals (CI). Results. Thirty-three trials (142 reports) were included (27 trials of sirolimus, 5 of everolimus, and 1 of head-to-head comparison). When TOR-I replaced calcineurin inhibitors (CNI) (8 trials with 750 participants), there was no difference in acute rejection (RR, 1.03; 95% CI, 0.74–1.44), but serum creatinine was lower (WMD, −18.31 &mgr;mol/L; 95% CI, −30.96 to −5.67) and bone marrow more suppressed (leukopenia: RR 2.02; 95% CI, 1.12–3.66; thrombocytopenia: RR, 6.97; 95% CI, 2.97–16.36; and anaemia: RR, 1.67; 95% CI, 1.27–2.20). When TOR-I replaced antimetabolites (11 trials with 3966 participants), acute rejection and cytomegalovirus infection (CMV) were reduced (RR, 0.84; 95% CI, 0.71–0.99; RR, 0.49; 95% CI, 0.37–0.65, respectively), but hypercholesterolemia was increased (RR, 1.65; 95% CI, 1.32–2.06). When low- was compared with high-dose TOR-I, with equal CNI dose (10 trials with 3,175 participants), rejection was increased (RR, 1.23; 95% CI, 1.06–1.43) but calculated glomerular filtration rate (GFR) higher (WMD, 4.27 mL/min; 95% CI, 1.12–7.41), and when lower-dose TOR-I and standard-dose CNI were compared with higher-dose TOR-I and reduced CNI, acute rejection was reduced (RR, 0.67; 95% CI, 0.52–0.88), but calculated GFR was also reduced (WMD, −9.46 mL/min; 95% CI, −12.16 to −6.76). There was no significant difference in mortality, graft loss, or malignancy risk for TOR-I in any comparison. Conclusions. TOR-I have been evaluated in four different primary immunosuppressive algorithms: as replacement for CNI and antimetabolites, in combination with CNI at low and high doses, and with a variable dose of CNI. Generally, surrogate endpoints for graft survival favor TOR-I (lower risk of acute rejection and higher GFR), and surrogate endpoints for patient outcomes are worsened by TOR-I (bone marrow suppression and lipid disturbance). Long-term hard-endpoint data from methodologically robust randomized trials are still required.

Adriamycin nephropathy: A model of focal segmental glomerulosclerosis

Adriamycin nephropathy (AN) is a rodent model of chronic kidney disease that has been studied extensively and has enabled a greater understanding of the processes underlying the progression of chronic proteinuric renal disease. AN is characterized by podocyte injury followed by glomerulosclerosis, tubulointerstitial inflammation and fibrosis. Genetic studies have demonstrated a number of loci that alter both risk and severity of renal injury induced by Adriamycin. Adriamycin‐induced renal injury has been shown in numerous studies to be modulated by both non‐immune and immune factors, and has facilitated further study of mechanisms of tubulointerstitial injury. This review will outline the pharmacological behaviour of Adriamycin, and describe in detail the model of AN, including its key structural characteristics, genetic susceptibility and pathogenesis.

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.

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.

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.

IL-25 Induces M2 Macrophages and Reduces Renal Injury in Proteinuric Kidney Disease

The kidney contains receptors for the cytokine IL-25, but the effects of IL-25 in CKD are unknown. Here, we induced adriamycin nephropathy in both BALB/c mice and severe combined immunodeficient (SCID) mice, and we injected IL-25 for 7 consecutive days starting at day 5 after adriamycin administration. BALB/c mice treated with IL-25 had less glomerulosclerosis, tubular atrophy, interstitial expansion, and proteinuria than control mice at day 28. IL-25 increased the levels of IL-4 and IL-13 in serum, kidney, renal draining lymph nodes, and CD4+ lymphocytes. IL-25 also directly suppressed effector macrophages in vitro and in vivo and induced alternatively activated (M2) macrophages in vivo. However, in SCID mice and in BALB/c mice treated with IL-4/13-neutralizing antibody, IL-25 failed to protect against renal injury and did not induce M2. In conclusion, IL-25 protects against renal injury in adriamycin nephropathy in mice by, at least in part, inducing Th2 immune responses.

Renal F4/80+CD11c+ Mononuclear Phagocytes Display Phenotypic and Functional Characteristics of Macrophages in Health and in Adriamycin Nephropathy

Conventional markers of macrophages (Mфs) and dendritic cells (DCs) lack specificity and often overlap, leading to confusion and controversy regarding the precise function of these cells in kidney and other diseases. This study aimed to identify the phenotype and function of renal mononuclear phagocytes (rMPs) expressing key markers of both Mфs and DCs. F4/80(+)CD11c(+) cells accounted for 45% of total rMPs in normal kidneys and in those from mice with Adriamycin nephropathy (AN). Despite expression of the DC marker CD11c, these double-positive rMPs displayed the features of Mфs, including Mф-like morphology, high expression of CD68, CD204, and CD206, and high phagocytic ability but low antigen-presenting ability. F4/80(+)CD11c(+) cells were found in the cortex but not in the medulla of the kidney. In AN, F4/80(+)CD11c(+) cells displayed an M1 Mф phenotype with high expression of inflammatory mediators and costimulatory factors. Adoptive transfer of F4/80(+)CD11c(+) cells separated from diseased kidney aggravated renal injury in AN mice. Furthermore, adoptive transfer of common progenitors revealed that kidney F4/80(+)CD11c(+) cells were derived predominantly from monocytes, but not from pre-DCs. In conclusion, renal F4/80(+)CD11c(+) cells are a major subset of rMPs and display Mф-like phenotypic and functional characteristics in health and in AN.