Renal fibrosis leads to end-stage renal disease demanding renal replacement therapy because no adequate treatment exists. IFN-g is an antifibrotic cytokine that may attenuate renal fibrosis. Systemically administered IFN-g causes side effects that may be prevented by specific drug targeting. Interstitial myofibroblasts are the effector cells in renal fibrogenesis. Here, we tested the hypothesis that cell-specific delivery of IFN-g to plateletderived growth factor receptor b (PDGFRb)-expressing myofibroblasts attenuates fibrosis in an obstructive nephropathy [unilateral ureteral obstruction (UUO)] mouse model. PEGylated IFN-g conjugated to PDGFRbrecognizing peptide [(PPB)-polyethylene glycol (PEG)-IFN-g] was tested in vitro and in vivo for antifibrotic properties and compared with free IFN-g. PDGFRb expression was >3-fold increased (P < 0.05) in mouse fibrotic UUO kidneys and colocalized with a-smooth muscle actin-positive (SMA + ) myofibroblasts.

Proteinuria is an important cause of progressive tubulo-interstitial damage. Whether proteinuria could trigger a renal lymphangiogenic response has not been established. Moreover, the temporal relationship between development of fibrosis, inflammation and lymphangiogenesis in chronic progressive kidney disease is not clear yet. Therefore, we evaluated the time course of lymph vessel (LV) formation in relation to proteinuria and interstitial damage in a rat model of chronic unilateral adriamycin nephrosis. Proteinuria and kidneys were evaluated up to 30 weeks after induction of nephrosis. LVs were identified by podoplanin/VEGFR3 double staining. After 6 weeks proteinuria was well-established, without influx of interstitial macrophages and myofibroblasts, collagen deposition, osteopontin expression (tubular activation) or LV formation. At 12 weeks, a ∼3-fold increase in cortical LV density was found (p<0.001), gradually increasing over time. This corresponded with a significant increase in tubular osteopontin expression (p<0.01) and interstitial myofibroblast numbers (p<0.05), whereas collagen deposition and macrophage numbers were not yet increased. VEGF-C was mostly expressed by tubular cells rather than interstitial cells. Cultured tubular cells stimulated with FCS showed a dose-dependent increase in mRNA and protein expression of VEGF-C which was not observed by human albumin stimulation. We conclude that chronic proteinuria provoked lymphangiogenesis in temporal conjunction with tubular osteopontin expression and influx of myofibroblasts, that preceded interstitial fibrosis.

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 (200-300 μm 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 TGFβ1 (5 ng/ml) for 48 h in the presence or absence of the anti-fibrotic cytokine IFNγ (1 µg/ml) or an IFNγ conjugate targeted to PDGFRβ (PPB-PEG-IFNγ). Following culture, tissue viability (ATP-content) and expression of α-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 TGFβ1 and IFNγ on viability were observed. TGFβ1 markedly increased α-SMA, fibronectin and collagen I mRNA and protein expression levels. IFNγ and PPB-PEG-IFNγ significantly reduced TGFβ1-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.