Effects of resin materials dedicated for additive manufacturing of temporary dental restorations on human gingival keratinocytes.

OBJECTIVE: This study investigated the effect of eluates of conventional and 3D-printed resin materials for manufacturing temporary dental restorations on gingival keratinocytes. METHODS: Three-dimensional (3D)-printed resin materials: 3Delta temp (Deltamed), NextDent MFH (Nextdent), Freeprint temp (Detax), GC temp (GC), were compared to Grandio disc (Voco) and Luxatemp (DMG). Human gingival keratinocytes (IHGKs) were exposed to eluates of the materials and XTT assays were performed at 24 h, 48 h, 72 h, or 144 h. For quantification of the proinflammatory response, the protein amount of IL-6 and 8 was determined in the supernatants using ELISA. One-way ANOVA with post hoc analysis was used to compare differences in cell viability and IL-6 and IL-8 levels between groups. RESULTS: At 24 h, and more remarkably at 48 h, a significant decrease in cell viability occurred for the 3D-printed materials compared to the untreated IHGKs, but also compared to Grandio disc and Luxatemp. Except for the expression of IL-8 in presence of the eluate of Grandio disc at 24 and 48 h, all tested materials caused attenuation of IL-6 and 8 from IHGKs for any observation period. CONCLUSIONS: The materials for additive manufacturing affect cell proliferation differently than the subtractive manufactured material Grandio disc and the conventional material Luxatemp. CLINICAL SIGNIFICANCE: In comparison to conventional and subtractive manufactured restorations, 3D printed temporary restorations might induce more negative effects on the gingival and probably also on pulpal health since viability and the proinflammatory response of oral keratinocytes are more intensively affected by these materials.

Transcriptome-based screening of ion channels and transporters in a migratory chondroprogenitor cell line isolated from late-stage osteoarthritic cartilage.

Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription-polymerase chain reaction, we examined the role of calcium-dependent potassium channels in CPCs and observed functional large-conductance calcium-activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca2+ events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in-depth characterisation of CPCs and their phenotype in the context of cartilage regeneration.

Donor variation and sex hormone receptors in periodontal ligament cells.

OBJECTIVE: This study evaluated the gene expression and protein synthesis of sex hormone receptors in human periodontal ligament cells (PDLCs) in relation to donor- and tooth-specific factors with the aim to clarify the debate about sex hormone receptors in PDLCs. DESIGN: The expression patterns of estrogen receptors (genes: ESR1 and ESR2; proteins: ERα and ERß), androgen receptor (AR) and progesterone receptor (PR) were investigated in the context of immortalization status, previous orthodontic tooth movement (OTM), donor age, sex and hormonal stimulation in PDLCs from 14 healthy donors (male: n = 8, female: n = 6; adolescents: n = 8, adults: n = 6) using quantitative real-time polymerase chain reaction, Western blot and immunocytochemistry. RESULTS: For ERß, the full-length isoform ERß1 and truncated variants were detected. For ERα, the expected isoform ERα66 was not observed, but a novel isoform ERα36 was detected. Immortalization status, previous OTM and donor age had no impact on ESR1 and ESR2 expression. Estradiol stimulation for 24 h doubled the ratio of ESR2/ESR1 in PDLCs from female but not male donors, indicating sex-specific patterns of receptor expression. AR and PR demonstrated insufficient protein synthesis in PDLCs. CONCLUSIONS: The data revealed a pivotal role for and complex interplay between ERα and ERß in human PDLCs regardless of variable donor characteristics. Therefore, PDLC biology might be altered in patients of each age group and both sexes due to hormonal changes. This should be kept in mind during periodontic and orthodontic treatment of patients with special hormonal status.

Decreased Expression of the Human Urea Transporter SLC14A1 in Bone is Induced by Cytokines and Stimulates Adipogenesis of Mesenchymal Progenitor Cells.

The human urea transporter SLC14A1 (HUT11/UT-B) has been suggested as a marker for the adipogenic differentiation of bone cells with a relevance for bone diseases. We investigated the function of SLC14A1 in different cells models from bone environment. SLC14A1 expression and cytokine production was investigated in bone cells obtained from patients with osteoporosis. Gene and protein expression of SLC14A1 was studied during adipogenic or osteogenic differentiation of human mesenchymal progenitor cells (hMSCs) and of the single-cell-derived hMSC line (SCP-1), as well as in osteoclasts and chondrocytes. Localization was determined by histochemical methods and functionality by urea transport experiments. Expression of SLC14A1 mRNA was lower in cells from patients with osteoporosis that produced high levels of cytokines. Accordingly, when adding a combination of cytokines to SCP-1 SLC14A1 mRNA expression decreased. SLC14A1 mRNA expression decreased after both osteogenic and more pronounced adipogenic stimulation of hMSCs and SCP-1 cells. The highest SLC14A1 expression was determined in undifferentiated cells, lowest in chondrocytes and osteoclasts. Downregulation of SLC14A1 by siRNA resulted in an increased expression of interleukin-6 and interleukin-1 beta as well as adipogenic markers. Urea influx through SLC14A1 increased expression of osteogenic markers, adipogenic markers were suppressed. SLC14A1 protein was localized in the cell membrane and the cytoplasm. Summarizing, the SLC14A1 urea transporter affects early differentiation of hMSCs by diminishing osteogenesis or by favoring adipogenesis, depending on its expression level. Therefore, SLC14A1 is not unequivocally an adipogenic marker in bone. Our findings suggest an involvement of SLC14A1 in bone metabolism and inflammatory processes and disease-dependent influences on its expression.

Molecular phenotyping of the surfaceome of migratory chondroprogenitors and mesenchymal stem cells using biotinylation, glycocapture and quantitative LC-MS/MS proteomic analysis.

The complement of cell surface proteins, collectively referred to as the surfaceome, is a useful indicator of normal differentiation processes, and the development of pathologies such as osteoarthritis (OA). We employed biochemical and proteomic tools to explore the surfaceome and to define biomarkers in chondrogenic progenitor cells (CPC) derived from human OA knee articular cartilage. These cells have great therapeutic potential, but their unexplored biology limits their clinical application. We performed biotinylation combined with glycocapture and high throughput shotgun proteomics to define the surface proteome of human bone marrow mesenchymal stem cells (MSCs) and human CPCs. We prepared cell surface protein-enriched fractions from MSCs and CPCs, and then a proteomic approach was used to compare and evaluate protein changes between undifferentiated MSCs and CPCs. 1256 proteins were identified in the study, of which 791 (63%) were plasma membrane, cell surface or extracellular matrix proteins. Proteins constituting the surfaceome were annotated and categorized. Our results provide, for the first time, a repository of quantitative proteomic data on the surfaceome of two closely related cell types relevant to cartilage biology and OA. These results may provide novel insights into the transformation of the surfaceome during chondrogenic differentiation and phenotypic changes during OA development.

The Influence of TGF-ß3, EGF, and BGN on SOX9 and RUNX2 Expression in Human Chondrogenic Progenitor Cells.

Osteoarthritis (OA) is the most common chronic joint disease and leads to the degradation of the extracellular matrix by an imbalance between anabolic and catabolic processes. TGF-ß3 (transforming growth factor beta-3) and epidermal growth factor (EGF) influence the osteochondrogenic potential of chondrocytes. In this study, we compared the expression of mediators and receptors in the TGF-ß3 and EGF pathways, as well as biglycan (BGN), in healthy and diseased chondrocytes. Furthermore, we used chondrogenic progenitor cells (CPCs) for in vitro stimulation and knockdown experiments to elucidate the effects of TGF-ß3 and EGF on the chondrogenic potential. Our results demonstrate that the expression of TGF-beta receptor type-1 (TGFBRI) and epidermal growth factor receptor (EGFR) is altered in diseased chondrocytes as well as in CPCs. Moreover, TGF-ß3 and EGF stimulation influenced the expression levels of BGN, SRY (sex determining region Y)-box 9 (SOX9), and Runt-related transcription factor 2 (RUNX2) in CPCs. Therefore, changes in TGFBRI and EGFR expression likely contribute to the degenerative and regenerative effects seen in late stages of OA.

Distinct functions of the dual leucine zipper kinase depending on its subcellular localization.

The dual leucine zipper kinase DLK induces ß-cell apoptosis by inhibiting the transcriptional activity conferred by the ß-cell protective transcription factor cAMP response element binding protein CREB. This action might contribute to ß-cell loss and ultimately diabetes. Within its kinase domain DLK shares high homology with the mixed lineage kinase (MLK) 3, which is activated by tumor necrosis factor (TNF) α and interleukin (IL)-1ß, known prediabetic signals. In the present study, the regulation of DLK in ß-cells by these cytokines was investigated. Both, TNFα and IL-1ß induced the nuclear translocation of DLK. Mutations within a putative nuclear localization signal (NLS) prevented basal and cytokine-induced nuclear localization of DLK and binding to the importin receptor importin α, thereby demonstrating a functional NLS within DLK. DLK NLS mutants were catalytically active as they phosphorylated their down-stream kinase c-Jun N-terminal kinase to the same extent as DLK wild-type but did neither inhibit CREB-dependent gene transcription nor transcription conferred by the promoter of the anti-apoptotic protein BCL-xL. In addition, the ß-cell apoptosis-inducing effect of DLK was severely diminished by mutation of its NLS. In a murine model of prediabetes, enhanced nuclear DLK was found. These data demonstrate that DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating DLK action. Furthermore, the prevention of the nuclear localization of DLK as induced by prediabetic signals with consecutive suppression of ß-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus.

Interleukin 17 inhibits progenitor cells in rheumatoid arthritis cartilage.

Mesenchymal stem cells are known to exert immunomodulatory effects in inflammatory diseases. Immuneregulatory cells lead to progressive joint destruction in rheumatoid arthritis (RA). Proinflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukins (ILs) are the main players. Here, we studied progenitor cells from RA cartilage (RA-CPCs) that are positive for IL-17 receptors to determinate the effects of inflammation on their chondrogenic potenial. IL-17A/F reduced the chondrogenic potential of these cells via the upregulation of RUNX2 protein and enhanced IL-6 protein and MMP3 mRNA levels. Blocking antibodies against IL-17 positively influenced their repair potential. Furthermore, treating the RA-CPCs with the anti-human IL-17 antibody secukinumab or the anti-TNF-α antibody adalimumab reduced the proinflammatory IL-6 protein level and positively influenced the secretion of anti-inflammatory IL-10 protein. Additionally, adalimumab and secukinumab in particular reduced RUNX2 protein to promote chondrogenesis. The amelioration of inflammation, particularly via IL-17 antagonism, might be a new therapeutic approach for enhancing intrinsic cartilage repair mechanisms in RA patients.

The growth plate's response to load is partially mediated by mechano-sensing via the chondrocytic primary cilium.

Mechanical load plays a significant role in bone and growth-plate development. Chondrocytes sense and respond to mechanical stimulation; however, the mechanisms by which those signals exert their effects are not fully understood. The primary cilium has been identified as a mechano-sensor in several cell types, including renal epithelial cells and endothelium, and accumulating evidence connects it to mechano-transduction in chondrocytes. In the growth plate, the primary cilium is involved in several regulatory pathways, such as the non-canonical Wnt and Indian Hedgehog. Moreover, it mediates cell shape, orientation, growth, and differentiation in the growth plate. In this work, we show that mechanical load enhances ciliogenesis in the growth plate. This leads to alterations in the expression and localization of key members of the Ihh-PTHrP loop resulting in decreased proliferation and an abnormal switch from proliferation to differentiation, together with abnormal chondrocyte morphology and organization. Moreover, we use the chondrogenic cell line ATDC5, a model for growth-plate chondrocytes, to understand the mechanisms mediating the participation of the primary cilium, and in particular KIF3A, in the cell's response to mechanical stimulation. We show that this key component of the cilium mediates gene expression in response to mechanical stimulation.

Purinergic signalling is required for calcium oscillations in migratory chondrogenic progenitor cells.

Osteoarthritis (OA) is the most common form of chronic musculoskeletal disorders. A migratory stem cell population termed chondrogenic progenitor cells (CPC) with in vitro chondrogenic potential was previously isolated from OA cartilage. Since intracellular Ca(2+) signalling is an important regulator of chondrogenesis, we aimed to provide a detailed understanding of the Ca(2+) homeostasis of CPCs. In this work, CPCs immortalised by lentiviral administration of the human telomerase reverse transcriptase (hTERT) and grown in monolayer cultures were studied. Expressions of all three IP3Rs were confirmed, but no RyR subtypes were detected. Ca(2+) oscillations observed in CPCs were predominantly dependent on Ca(2+) release and store replenishment via store-operated Ca(2+) entry; CPCs express both STIM1 and Orai1 proteins. Expressions of adenosine receptor mRNAs were verified, and adenosine elicited Ca(2+) transients. Various P2 receptor subtypes were identified; P2Y1 can bind ADP; P2Y4 is targeted by UTP; and ATP may evoke Ca(2+) transients via detected P2X subtypes, as well as P2Y1 and P2Y2. Enzymatic breakdown of extracellular nucleotides by apyrase completely abrogated Ca(2+) oscillations, suggesting that an autocrine/paracrine purinergic mechanism may drive Ca(2+) oscillations in these cells. As CPCs possess a broad spectrum of functional molecular elements of Ca(2+) signalling, Ca(2+)-dependent regulatory mechanisms can be supposed to influence their differentiation potential.

Human migratory meniscus progenitor cells are controlled via the TGF-ß pathway.

Degeneration of the knee joint during osteoarthritis often begins with meniscal lesions. Meniscectomy, previously performed extensively after meniscal injury, is now obsolete because of the inevitable osteoarthritis that occurs following this procedure. Clinically, meniscus self-renewal is well documented as long as the outer, vascularized meniscal ring remains intact. In contrast, regeneration of the inner, avascular meniscus does not occur. Here, we show that cartilage tissue harvested from the avascular inner human meniscus during the late stages of osteoarthritis harbors a unique progenitor cell population. These meniscus progenitor cells (MPCs) are clonogenic and multipotent and exhibit migratory activity. We also determined that MPCs are likely to be controlled by canonical transforming growth factor ß (TGF-ß) signaling that leads to an increase in SOX9 and a decrease in RUNX2, thereby enhancing the chondrogenic potential of MPC. Therefore, our work is relevant for the development of novel cell biological, regenerative therapies for meniscus repair.

Cartilage repair in vivo: the role of migratory progenitor cells.

The most common diseases of the joints and its tissues are osteoarthritis and rheumatoid arthritis, with osteoarthritis being anticipated to be the fourth leading cause of disability by the year 2020. To date, no truly causal therapies are available, and this has promoted tissue engineering attempts mainly involving mesenchymal stem cells. The goal of all tissue repairs would be to restore a fully functional tissue, here a hyaline articular cartilage. The hyaline cartilage is the most affected in osteoarthritis, where altered cell-matrix interactions gradually destroy tissue integrity. In rheumatoid arthritis, the inflammatory aspect is more important, and the cartilage tissue is destroyed by the invasion of tumor-like pannus tissue arising from the inflamed synovia. Furthermore, the fibrocartilage of the meniscus is clearly involved in the initiation of osteoarthritis, especially after trauma. Recent investigations have highlighted the role of migratory progenitor cells found in diseased tissues in situ. In osteoarthritis and rheumatoid arthritis, these chondrogenic progenitor cells are involved in regeneration efforts that are largely unsuccessful in diseased cartilage tissue. However, these progenitor cells are interesting targets for a cell-based regenerative therapy for joint diseases.

Norepinephrine inhibition of mesenchymal stem cell and chondrogenic progenitor cell chondrogenesis and acceleration of chondrogenic hypertrophy.

OBJECTIVE: Mesenchymal progenitor cell chondrogenesis is the biologic platform for the generation or regeneration of cartilage, but the external influence of the sympathetic nervous system on this process is not yet known. Sympathetic nerve fibers are present in articular tissue, and the sympathetic nervous system influences the musculoskeletal system by, for example, increasing osteoclastogenesis. This study was initiated to explore the role of the sympathetic neurotransmitter norepinephrine (NE) in mesenchymal stem cell (MSC)-dependent and cartilage progenitor cell (CPC)-dependent chondrogenesis. METHODS: Using human MSCs or CPCs, chondrogenic differentiation was induced in the presence of NE, the specific ß-adrenergic receptor (ß-AR) agonist isoproterenol, and the specific ß-AR antagonist nadolol. We studied sympathetic nerve fibers, tyrosine hydroxylase (TH) expression, catecholamine biosynthesis, and synovial fluid levels in human joints, as well as cartilage-specific matrix deposition during differentiation. RESULTS: TH+ sympathetic nerve fibers were present in the synovial tissue, meniscus, and subchondral bone marrow. In addition, synovial fluid from patients with knee trauma demonstrated high concentrations of NE. During MSC or CPC chondrogenesis, ß-AR were expressed. Chondrogenic aggregates treated with NE or isoproterenol synthesized lower amounts of type II collagen and glycosaminoglycans. NE and isoproterenol treatment dose-dependently increased the levels of cartilage hypertrophy markers (type X collagen and matrix metalloproteinase 13). Nadolol reversed the inhibition of chondrogenesis and the up-regulation of cartilage hypertrophy. CONCLUSION: Our findings demonstrate NE-dependent inhibition of chondrogenesis and acceleration of hypertrophic differentiation. By inhibiting cartilage repair, these sympathetic influences can be important after joint trauma. These findings may be a basis for novel neurochondrogenic therapeutic options.

A discoidin domain receptor 1 knock-out mouse as a novel model for osteoarthritis of the temporomandibular joint.

Discoidin domain receptor 1 (DDR-1)-deficient mice exhibited a high incidence of osteoarthritis (OA) in the temporomandibular joint (TMJ) as early as 9 weeks of age. They showed typical histological signs of OA, including surface fissures, loss of proteoglycans, chondrocyte cluster formation, collagen type I upregulation, and atypical collagen fibril arrangements. Chondrocytes isolated from the TMJs of DDR-1-deficient mice maintained their osteoarthritic characteristics when placed in culture. They expressed high levels of runx-2 and collagen type I, as well as low levels of sox-9 and aggrecan. The expression of DDR-2, a key factor in OA, was increased. DDR-1-deficient chondrocytes from the TMJ were positively influenced towards chondrogenesis by a three-dimensional matrix combined with a runx-2 knockdown or stimulation with extracellular matrix components, such as nidogen-2. Therefore, the DDR-1 knock-out mouse can serve as a novel model for temporomandibular disorders, such as OA of the TMJ, and will help to develop new treatment options, particularly those involving tissue regeneration.

Current concepts in stem cell therapy for articular cartilage repair.

INTRODUCTION: Hyaline articular cartilage is the connective tissue responsible for frictionless joint movement. Its degeneration ultimately results in complete loss of joint function in the late stages of osteoarthritis. Intrinsic repair is compromised, and cartilage tissue regeneration is difficult. However, new options are available to repair cartilage tissue by applying ESCs, MSCs and CPCs. AREAS COVERED: In this review, the authors shed light on the different concepts currently under investigation for cartilage repair. EXPERT OPINION: So far, there is no way to derive a chondrogenic lineage from stem cells that forms functional hyaline cartilage tissue in vivo. One alternative might be to enhance the chondrogenic potential of repair cells, which are already present in diseased cartilage tissue. CPCs found in diseased cartilage tissue in situ are biologically driven toward the osteochondrogenic lineage and can be directed toward chondrogenesis at least in vitro.

Plasma leakage through glomerular basement membrane ruptures triggers the proliferation of parietal epithelial cells and crescent formation in non-inflammatory glomerular injury.

Glomerular crescents are most common in rapidly progressive glomerulonephritis but also occur in non-inflammatory chronic glomerulopathies; thus, factors other than inflammation should trigger crescent formation, eg vascular damage and plasma leakage. Here we report that Alport nephropathy in Col4A3-deficient Sv129 mice is complicated by diffuse and global crescent formation in which proliferating parietal epithelial cells are the predominant cell type. Laminin staining and transmission and acellular scanning electron microscopy of acellular glomeruli documented disruptions and progressive disintegration of the glomerular basement membrane in Col4A3-deficient mice. FITC-dextran perfusion further revealed vascular leakage from glomerular capillaries into Bowman's space, further documented by fibrin deposits in the segmental crescents. Its pathogenic role was validated by showing that the fibrinolytic activity of recombinant urokinase partially prevented crescent formation. In addition, in vitro studies confirmed an additional mitogenic potential of serum on murine and human parietal epithelial cells. Furthermore, loss of parietal cell polarity and unpolarized secretion of extracellular matrix components were evident within fibrocellular crescents. Among 665 human Alport nephropathy biopsies, crescent formation was noted in 0.4%. We conclude that glomerular vascular injury and GBM breaks cause plasma leakage which triggers a wound healing programme involving the proliferation of parietal cells and their loss of polarity. This process can trigger cellular and fibrocellular crescent formation even in the absence of cellular inflammation and rupture of the Bowman's capsule.

New insights into cartilage repair - the role of migratory progenitor cells in osteoarthritis.

Osteoarthritis is one of the most common musculo-skeletal diseases with a complex patholoy and a strong impact on cell biology, differentiation and migration behavior of mesenchymal stem cell-derived progenitor cells. In this review, we elucidate the influence of the pathologically altered extracellular matrix on progenitor cell behavior. Moreover, we discuss the modulation of progenitor cells especially of previously characterized chondrogenic progenitor cells (Koelling et al., 2009) in situ to enhance their regeneration potential. These options comprise the application of growth factors like fibroblast growth factor-2, a Runx-2 knock down and a contemporary anti-inflammatory therapy. This supports endogenous regeneration on behalf of the diseased osteoarthritic cartilage, which otherwise results mainly in an insufficient fibro-cartilaginous repair tissue. Furthermore, new results indicate a role of pericytes in osteoarthritis for these repair attempts. We discuss the biological mechanisms potentially leading to new therapeutic options in osteoarthritis to enhance regeneration in situ.

Absence of the basement membrane component nidogen 2, but not of nidogen 1, results in increased lung metastasis in mice.

Nidogen 1 and 2 are ubiquitous basement membrane (BM) components. They show a divergent expression pattern in certain adult tissues with a prominent localization of nidogen 2 in blood vessel BMs. Deletion of either nidogen 1 or 2 in mice had no effect on BM formation, suggesting complementary functions. However, studies in these mice revealed isoform-specific functions with nidogen 1-deficient mice showing neurological abnormalities and wound-healing defects not seen in the absence of nidogen 2. To investigate this further nidogen 1- or 2-deficient mice were intravenously injected with B16 murine melanoma cells, and lung metastasis was analyzed. The authors could show that loss of nidogen 2, but not of nidogen 1, significantly promotes lung metastasis of melanoma cells. Histological and ultrastructural analysis of nidogen 1- and 2-deficient lungs did not reveal differences in morphology and ultrastructure of BMs, including vessel BMs. Furthermore, deposition and distribution of the major BM components were indistinguishable between the two mouse strains. Taken together, these results suggest that absence of nidogen 2 might result in subtle changes of endothelial BMs in the lung, which would allow faster passage of tumor cells through these BMs, leading to a higher metastasis rate and more larger tumors.

Tumour necrosis factor-α drives Alport glomerulosclerosis in mice by promoting podocyte apoptosis.

Chronic renal failure involves the progressive loss of renal parenchymal cells. For example, Alport syndrome develops from mutated type IV collagen that fosters the digestion of glomerular basement membranes and podocyte loss, followed by progressive glomerulosclerosis, ie Alport nephropathy. Here we show that autosomal recessive Alport nephropathy in collagen 4a3-deficient mice is associated with increased intrarenal expression of the pro-apoptotic cytokine tumour necrosis factor-alpha (TNF-α) in glomerular cells including podocytes as well as in infiltrating leukocytes. We therefore hypothesized that TNF-α contributes to Alport glomerulosclerosis by inducing podocyte apoptosis. To address this issue, we treated 4-week-old collagen 4a3-deficient mice with either vehicle or the TNF-α antagonist etanercept for a period of 5 weeks. Etanercept treatment prolonged mean survival from 68 to 81 days as compared to vehicle-treated mice. The beneficial effect of etanercept on survival was associated with a significant improvement of the glomerulosclerosis score, proteinuria, and the glomerular filtration rate at 9 weeks of age. Etanercept treatment specifically reduced the numbers of apoptotic podocytes, increased total podocyte counts, and increased the renal mRNA expression of nephrin and podocin without affecting markers of renal inflammation. TNF-α-induced podocyte loss is a previously unrecognized pathological mechanism of Alport glomerulosclerosis, and TNF-α blockade might be a therapeutic option to delay the progression of Alport nephropathy and potentially of other forms of glomerulosclerosis.

Bacterial CpG-DNA accelerates Alport glomerulosclerosis by inducing an M1 macrophage phenotype and tumor necrosis factor-α-mediated podocyte loss.

Loss of function mutations in the α3 or α4 chain of type IV collagen cause Alport nephropathy, characterized by progressive glomerulosclerosis. While studying the mechanisms that determine disease progression, we found that the evolution of kidney disease in Col4a3-deficient mice was associated with an influx of immune cell subsets including nonactivated macrophages. This suggested that intrarenal inflammation might accelerate Alport nephropathy. A possible mechanism might be the well-known enhancement of immune recognition by bacterial products. We found that exposure to bacterial endotoxin from 4 to 6 weeks of age did not affect disease progression, whereas an equipotent dose of cytosine-guanine (CpG)-DNA, a synthetic mimic of bacterial DNA, accelerated all aspects of Alport nephropathy and reduced the overall lifespan of Col4a3-deficient mice. This effect was associated with a significant increase of renal CD11b+/Ly6C(hi) macrophages, intrarenal production of inducible nitric oxide synthase, tumor necrosis factor (TNF)-α, interleukin-12, and CXCL10, and loss of podocytes. TNF-α was essential for acceleration of Alport nephropathy, as etanercept (a soluble TNF-α receptor) entirely abrogated the CpG-DNA effect. Thus, systemic exposure to CpG-DNA induces classically activated (M1) macrophages that enhance intrarenal inflammation and disease progression. Hence, factors that modulate the phenotype of renal macrophages can affect the progression of Alport nephropathy and, potentially, other types of chronic kidney diseases.