Transgene therapy for rat anti-Thy1.1 glomerulonephritis via mesangial cell vector with a polyethylenimine/decorin nanocomplex

Abstract

Polyethylenimine (PEI), a cationic polymer, is one of the most efficient non-viral vectors for transgene therapy. Decorin (DCN), a leucine-rich proteoglycan secreted by glomerular mesangial cells (MC), is a promising anti-fibrotic agent for the treatment of glomerulonephritis. In this study, we used PEI-DCN nanocomplexes with different N/P ratios to transfect MC in vitro and deliver the MC vector with PEI-DCN expressing into rat anti-Thy1.1 nephritis kidney tissue via injection into the left renal artery in vivo. The PEI-plasmid DNA complex at N/P 20 had the highest level of transfection efficiency and the lowest level of cytotoxicity in cultured MC. Following injection, the ex vivo gene was transferred successfully into the glomeruli of the rat anti-Thy1.1 nephritis model by the MC vector with the PEI-DCN complex. The exogenous MC with DCN expression was located mainly in the mesangium and the glomerular capillary. Over-expression of DCN in diseased glomeruli could result in the inhibition of collagen IV deposition and MC proliferation. The pathological changes of rat nephritis were alleviated following injection of the vector. These findings demonstrate that the DCN gene delivered by the PEI-DNA nanocomplex with the MC vector is a promising therapeutic method for the treatment of glomerulonephritis.

Figure 1

Particle size (A) and zeta potential (B) of PEI–pDNA complexes at different N/P ratios. Data are expressed as the mean and standard error of at least three separate measurements.

Figure 2

Evaluation of the transfection efficiency in MC with PEI–pEGFP complexes.A, Fluorescence micrograph of MC transfected with PEI–pEGFP complexes for 48 h at different N/P ratios. (a) MC were transfected with Lipofectamine™ 2000 as a positive control. (b) N/P 1, (c) N/P 5, (d) N/P 10, (e) N/P 15 and (f) N/P 20. (×100) B, The transfection efficiency of MC with different N/P ratios was evaluated by flow cytometry. Cells treated with naked plasmid were used as a negative control. C, Cytotoxicity analysis of MC with different N/P ratios by the MTT test. Cells treated with naked plasmid were used as a negative control and cells transfected with Lipofectamine™ 2000 were used as a positive control. *P <0.01 vs control. The results are representative of at least three similar experiments.

Figure 3

DCN over-expression inhibited expression of TGF-β1 and collagen IV in MC.A, The protein level of DCN was assessed by Western blotting in PEI–pDCN transfected MC. Normal mesangial cells were used as a control. D1–D6 are different cell clones. B, The relative levels of DCN, TGF-β1 and collagen IV protein were determined by Western blotting in D6. Lanes 1, normal mesangial cells; 2, pcDNA3.1A-transfected MC; and 3, DCN-transfected MC (D6). Data are presented as mean ± SD. *P <0.01 vs MC and MC/pcDNA3.1A. The experiment was done at least three times.

Figure 4

Location of PEI–pEGFP or PEI–DCN in injected glomeruli. At 48 h after injection of the MC vector with PEI–pEGFP or PEI–DCN, paraffin sections of kidney were prepared and examined by immunohistochemistry. (A) Normal control glomerulus negative for EGFP staining. (B) Glomerulus of injected kidney with PEI–pEGFP expressing MC, the results show EGFP-positive cells are located mainly in the mesangial area and a few epithelial cells of the proximal tubule. (a,b ABC, ×100) (C) Glomerulus of injected kidney with PEI–DCN expressing MC. The exogenous DCN-positive cells have the same localization as EGFP in glomeruli. (D) Glomerulus of injected kidney with PEI–DCN expressing MC. The vimentin-positive MC are distributed mainly in the mesangium of glomeruli, which is consistent with the distribution pattern of PEI-DCN positive cells (c,d ABC × 200).

Figure 5

Immunohistochemistry results of DCN, collage IV and PCNA in rat Thy1.1 glomerulonephritis. Immunostaining of (A) DCN, (B)collage IV and (C) PCNA in rat Thy1.1 glomerulonephritis. (a–d) DCN expression in glomerulus of uninjected kidney. (e–h) DCN-positive distribution in glomerulus 1, 3 and 5 days after injection with PEI–DCN. Level of collagen IV expression in glomerulus of (i–l) uninjected kidney and (m–p) injected kidney. PCNA protein level in glomerulus of (q–t) uninjected kidney and (u–x) injected kidney. (ABC × 200). B, Immunohistochemistry results and statistical analysis. ⋆, P <0.05 vs uninjected side; *, P <0.01 vs uninjected side; ▴, P <0.05 vs control; ♦, P <0.01 vs control.