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.2016 May 22:11:77-84.
doi: 10.4137/BMI.S38439. eCollection 2016.

Precision-Cut Kidney Slices as a Tool to Understand the Dynamics of Extracellular Matrix Remodeling in Renal Fibrosis

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Precision-Cut Kidney Slices as a Tool to Understand the Dynamics of Extracellular Matrix Remodeling in Renal Fibrosis

Federica Genovese et al. Biomark Insights..

Abstract

The aim of this study was to set up an ex vivo model for renal interstitial fibrosis in order to investigate the extracellular matrix (ECM) turnover profile in the fibrotic kidney. We induced kidney fibrosis in fourteen 12-week-old male Sprague Dawley rats by unilateral ureteral obstruction (UUO) surgery of the right ureter. The left kidney (contralateral) was used as internal control. Six rats were sham operated and used as the control group. Rats were terminated two weeks after the surgery; the kidneys were excised and precision-cut kidney slices (PCKSs) were cultured for five days in serum-free medium. Markers of collagen type I formation (P1NP), collagen type I and III degradation (C1M and C3M), and α-smooth muscle actin (αSMA) were measured in the PCKS supernatants by enzyme-linked immunosorbent assay. P1NP, C1M, C3M, and α-SMA were increased up to 2- to 13-fold in supernatants of tissue slices from the UUO-ligated kidneys compared with the contralateral kidneys (P < 0.001) and with the kidneys of sham-operated animals (P < 0.0001). The markers could also reflect the level of fibrosis in different animals. The UUO PCKS ex vivo model provides a valuable translational tool for investigating the extracellular matrix remodeling associated with renal interstitial fibrosis.

Keywords: PCKS; UUO; ex vivo; extracellular matrix; kidney fibrosis.

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Figures

Figure 1
Figure 1
(A) Weight of excised kidneys at termination. The higher weight of obstructed kidneys was due to both enlargement of the kidney and the presence of excessive accumulated liquid in the tissue. (B) Tissue viability of PCKSs after 5 days of culture (% AlamarBlue® fluorescence compared with day 0). (C) Levels of active TGF-β in PCKS from UUO right and left kidneys and control right kidneys. Statistical difference calculated with parametrict-test (*P < 0.05;**P < 0.01;****P < 0.0001), pairedt-test (###P < 0.001), or Wilcoxon test ($$$$P < 0.0001).
Figure 2
Figure 2
Concentration of ECM remodeling markers in supernatants of PCKS. Levels of P1NP (formation of collagen type I) (A), C1M (MMP-mediated degradation of collagen type I) (B), C3M (MMP-mediated degradation of collagen type III) (C), and α-SMA (D) in PCKS from UUO-operated animals (right kidney: obstructed kidney; left kidney: contralateral kidney) and control sham-operated animals (sham).Notes: Statistical difference calculated with Wilcoxon test (***P < 0.001) or Mann–Whitney test (##P < 0.01;####P < 0.0001). Data presented as mean ± SEM.
Figure 3
Figure 3
(A) % level of markers in supernatant of PCKS from the right kidney (the obstructed one in UUO-operated animal) compared with the left kidney in individual animals. (B) Representative sections of right kidneys from five UUO-operated animals (#1, #7, #8, #11, and #15) and a sham-operated animal (#19), stained with Masson’s trichrome staining.
Figure 4
Figure 4
Correlation plot of degree of fibrosis (evaluated as the mean of the % of blue staining on three representative pictures of each slide stained with Masson’s trichrome staining) in the right kidney of different animals with marker levels in tissue supernatant.
Figure 5
Figure 5
α-SMA immunohistochemistry on two representative UUO-operated animals (#7 and #11) and a representative sham-operated animal (#19). The left kidney is the contralateral kidney. The α-SMA staining in the operated right kidney colocalize with the collagen deposition in the Masson’s trichrome staining, in blue.
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