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

Abstract

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.

siRNA-Mediated RNA Interference in Precision-Cut Tissue Slices Prepared from Mouse Lung and Kidney

“…However, both untransfected and transfected PCKS contained some apoptotic proximal tubules (marked by loss of nuclei and swelling of cells), although the general morphology remained unaffected. Apoptosis in proximal tubules has been reported previously, and the authors suggested it is caused by a disturbed pre-urine flow [8,22]. As such, observed morphological changes in PCKS are not attributable to the siRNA.…”

“…Furthermore, the greater number of cells per unit volume in PCKS might have also affected the penetration of Accell siRNA by restraining the diffusion of siRNA from a region of high concentration to a region of low concentration. Researchers have previously shown that fluorescent lysozyme (~15 kDa) diffused into PCKS prepared from mice [21,22]. As such, it is not surprising that Accell siRNA, which has a comparable molecular weight to the fluorescent lysozyme, could also diffuse into PCKS.…”

Molecular validation of the precision-cut kidney slice (PCKS) model of renal fibrosis through assessment of TGF-β1-induced Smad and p38/ERK signaling

“…The PCKS model was constructed according to Potti et al's protocol [3]. Male C57BL/6 mice (weight: 25-30 g) were purchased from Beijing Vital River Laboratories (Beijing, China).…”

“…Although in vitro cell culture models have bettered our understanding of renal fibrosis [2], these models cannot adequately mimic the in vivo environment [3]. To address the limitation, a recent 2015 study by Poosti et al constructed an ex vivo precision-cut kidney slice (PCKS) model with the aim of mimicking renal fibrosis [3].…”

“…To address the limitation, a recent 2015 study by Poosti et al constructed an ex vivo precision-cut kidney slice (PCKS) model with the aim of mimicking renal fibrosis [3]. Specifically, PCKS prepared from healthy C57BL/6 mouse kidneys were incubated with transforming growth factor-β1 (TGF-β1) for 48 h to induce changes mimicking the renal fibrotic process (namely, increased α-smooth muscle actin (α-SMA), fibronectin, and collagen I levels) [3]. On the basis of Poosti et al's findings, the PCKS model appears to be a useful ex vivo model for investigating the pathoetiology of renal fibrosis and screening the efficacy of anti-fibrotic therapies.…”