Cell-specific targeting of renal fibrosis

De focus van dit proefschrift ligt op het gunstige effect zien van celspecifieke getargete afgifte om transplantaatafstoting, ontsteking en fibrose te verbeteren in diermodellen van nierziekten. We bieden een nieuwe strategie van medicamentafgifte aan proximale tubuluscellen door gebruik van de megalinerecptor en aan myofibroblasten door antifibrotische cytokines naar PDGFβR te dirigeren. Deze strategieën vergroten het therapeutische potentieel van medicamenten door verhoogde doeltreffendheid en verminderde off-target bijwerkingen. We geloven dat celspecifieke afgifte van medicamenten een veelbelovende aanpak is om effectief renale fibrose te stoppen. Bovendien is het ex vivo-model dat we hier beschreven hebben een nieuwe methode om de pathofysiologie van het vroege proces en het eindstadium van fibrose te bestuderen, evenals om te screenen op de werkzaamheid van antifibrotische medicamenten ex vivo in een multicellulair en profibrotisch milieu. Belangrijke voordelen van het slice-model is dat de data van onze dierproef direct geëxtrapoleerd kunnen worden naar de humane situatie door het gebruik van (fibrotisch) humaan nierweefsel. Van belang is dat weefselslices een alternatief, of 3V-methode, vormen die inzicht verschaffen in mechanismen van ziekteprocessen en ontworpen zijn om dierexperimenten te vervangen, te verminderen en te verfijnen

Targeting tubulointerstitial remodeling in proteinuric nephropathy in rats

Proteinuria is an important cause of tubulointerstitial damage. Anti-proteinuric interventions are not always successful, and residual proteinuria often leads to renal failure. This indicates the need for additional treatment modalities by targeting the harmful downstream consequences of proteinuria. We previously showed that proteinuria triggers renal lymphangiogenesis before the onset of interstitial inflammation and fibrosis. However, the interrelationship of these interstitial events in proteinuria is not yet clear. To this end, we specifically blocked lymphangiogenesis (anti-VEGFR3 antibody), monocyte/macrophage influx (clodronate liposomes) or lymphocyte and myofibroblast influx (S1P agonist FTY720) separately in a rat model to investigate the role and the possible interaction of each of these phenomena in tubulointerstitial remodeling in proteinuric nephropathy. Proteinuria was induced in 3-month old male Wistar rats by adriamycin injection. After 6 weeks, when proteinuria has developed, rats were treated for another 6 weeks by anti-VEGFR3 antibody, clodronate liposomes or FTY720 up to week 12. In proteinuric rats, lymphangiogenesis, influx of macrophages, T cells and myofibroblasts, and collagen III deposition and interstitial fibrosis significantly increased at week 12 vs week 6. Anti-VEGFR3 antibody prevented lymphangiogenesis in proteinuric rats, however, without significant effects on inflammatory and fibrotic markers or proteinuria. Clodronate liposomes inhibited macrophage influx and partly reduced myofibroblast expression; however, neither significantly prevented the development of lymphangiogenesis, nor fibrotic markers and proteinuria. FTY720 prevented myofibroblast accumulation, T-cell influx and interstitial fibrosis, and partially reduced macrophage number and proteinuria; however, it did not significantly influence lymphangiogenesis and collagen III deposition. This study showed that proteinuria-induced interstitial fibrosis cannot be halted by blocking lymphangiogenesis or the influx of macrophages. On the other hand, FTY720 treatment did prevent T-cell influx, myofibroblast accumulation and interstitial fibrosis, but not renal lymphangiogenesis and proteinuria. We conclude that tubulointerstitial fibrosis and inflammation are separate from lymphangiogenesis, at least under proteinuric conditions

Targeted Renal Delivery of Protein Kinase Inhibitors for the Treatment of Chronic Kidney Disease

The increasing prevalence of end-stage renal disease urges novel therapeutic strategies for the treatment of chronic kidney disease. As protein kinases play a pivotal role in renal inflammation and fibrosis, specific protein kinase inhibitors have been demonstrated to be renoprotective in experimental studies. However, since protein kinases are also involved in key physiological mechanisms such as cell differentiation, cell growth and proliferation, these beneficial effects have been associated with serious side effects, limiting their clinical applicability. However, the possibility to selectively deliver a drug to cells with a particular phenotype (i.e. cells expressing a cell-specific protein to which drugs can be targeted) has increased the potential of protein kinase inhibitors in chronic kidney disease. Several studies have reported renoprotective effects of protein kinase inhibitors specifically delivered to fibrotic cells, or to specific cell types such as proximal tubular epithelial cells or mesangial cells. An overview of these studies will be provided, as well as future directions in this exciting field of research that may lead to novel highly specific pharmacological intervention strategies

Precision-cut kidney slices (PCKS) to study development of renal fibrosis and efficacy of drug targeting ex vivo

Renal fibrosis is a serious clinical problem resulting in the greatest need for renal replacement therapy. No adequate preventive or curative therapy is available that could be clinically used to target renal fibrosis specifically. The search for new efficacious treatment strategies is therefore warranted. Although in vitro models using homogeneous cell populations have contributed to the understanding of the pathogenetic mechanisms involved in renal fibrosis, these models poorly mimic the complex in vivo milieu. Therefore, we here evaluated a precision-cut kidney slice (PCKS) model as a new, multicellular ex vivo model to study the development of fibrosis and its prevention using anti-fibrotic compounds. Precision-cut slices (200300 mu m thickness) were prepared from healthy C57BL/6 mouse kidneys using a Krumdieck tissue slicer. To induce changes mimicking the fibrotic process, slices were incubated with TGF beta 1 (5 ng/ml) for 48 h in the presence or absence of the anti-fibrotic cytokine IFN gamma (1 mu g/ml) or an IFN gamma conjugate targeted to PDGFR beta (PPB-PEG-IFN gamma). Following culture, tissue viability (ATP-content) and expression of alpha-SMA, fibronectin, collagen I and collagen III were determined using real-time PCR and immunohistochemistry. Slices remained viable up to 72 h of incubation, and no significant effects of TGF beta 1 and IFN gamma on viability were observed. TGF beta 1 markedly increased alpha-SMA, fibronectin and collagen I mRNA and protein expression levels. IFN gamma and PPB-PEG-IFN gamma significantly reduced TGF beta 1-induced fibronectin, collagen I and collagen III mRNA expression, which was confirmed by immunohistochemistry. The PKCS model is a novel tool to test the pathophysiology of fibrosis and to screen the efficacy of anti-fibrotic drugs ex vivo in a multicellular and pro-fibrotic milieu. A major advantage of the slice model is that it can be used not only for animal but also for (fibrotic) human kidney tissue

The GAG-Binding Peptide MIG30 Protects against Liver Ischemia-Reperfusion in Mice

Ischemia-reperfusion injury (IRI) drives graft rejection and is the main cause of mortality after liver transplantation. During IRI, an intense inflammatory response marked by chemokine production and neutrophil recruitment occurs. However, few strategies are available to restrain this excessive response. Here, we aimed to interfere with chemokine function during IRI in order to disrupt neutrophil recruitment to the injured liver. For this, we utilized a potent glycosaminoglycan (GAG)-binding peptide containing the 30 C-terminal amino acids of CXCL9 (MIG30) that is able to inhibit the binding of chemokines to GAGs in vitro. We observed that mice subjected to IRI and treated with MIG30 presented significantly lower liver injury and dysfunction as compared to vehicle-treated mice. Moreover, the levels of chemokines CXCL1, CXCL2 and CXCL6 and of proinflammatory cytokines TNF-α and IL-6 were significantly reduced in MIG30-treated mice. These events were associated with a marked inhibition of neutrophil recruitment to the liver during IRI. Lastly, we observed that MIG30 is unable to affect leukocytes directly nor to alter the stimulation by either CXCL8 or lipopolysaccharide (LPS), suggesting that its protective properties derive from its ability to inhibit chemokine activity in vivo. We conclude that MIG30 holds promise as a strategy to treat liver IRI and inflammation

Reduction of myeloid-derived suppressor cells reinforces the anti-solid tumor effect of recipient leukocyte infusion in murine neuroblastoma-bearing allogeneic bone marrow chimeras

Allogeneic hematopoietic stem cell transplantation is an emerging treatment option for solid tumors because of its capacity to elicit immune graft-versus-tumor effects. However, these are often limited and associated with GvHD. Adoptive recipient leukocyte infusion (RLI) was shown to enhance anti-tumor responses of allogeneic bone marrow transplantation in murine neuroblastoma (Neuro2A)-bearing chimeras. In contrast to the clinically used donor leukocyte infusion, the RLI anti-tumor effect-elicited by host-versus-graft lymphohematopoietic reactivity-does not cause GvHD; however, the tumor growth-inhibitory effect is incomplete, because overall survival is not prolonged. Here, we studied the anti-solid tumor mechanisms of RLI with the objective to improve its efficacy. Host-versus-graft reactivity following RLI was associated with a systemic cytokine storm, lymph node DC activation, and systemic expansion of host-derived IFN-γ-expressing CD4+ T cells and IFN-γ-and granzyme B-expressing CD8+ T cells, which acquired killing activity against Neuro2A and third-party tumor cells. The tumor showed up-regulation of MHC class I and a transient accumulation of IFN-γ-and granzyme B-expressing CD8+ T cells: the intra-tumor decline in cytotoxic CD8+ T cells coincided with a systemic-and to a lesser extent intra-tumoral-expansion of MDSC. In vivo MDSC depletion with 5-FU significantly improved the local tumor growth-inhibitory effect of RLI as well as overall survival. In conclusion, the RLI-induced alloreactivity gives rise to a host-derived cytotoxic T-cell anti-neuroblastoma response, but also drives an expansion of host-type MDSC that counteracts the anti-tumor effect. This finding identifies MDSC as a novel target to increase the effectiveness of RLI, and possibly other cancer immunotherapies.status: publishe