Low‐dose paclitaxel ameliorates fibrosis in the remnant kidney model by down‐regulating miR‐192

Abstract

Transforming growth factor (TGF)-β has been shown to play a central role in the development of tubulointerstitial fibrosis, which can be corrected via treatment with paclitaxel. The biology of microRNA (miR) can be modulated by paclitaxel. We hypothesized that paclitaxel may attenuate renal fibrosis in a rat model of remnant kidney disease by inhibiting TGF-β induced-miRs. Rats in groups of 12 were subjected to 5/6 nephrectomy and received low-dose intraperitoneal injection of paclitaxel. Renal functions were assessed at 8 weeks. The TGF-β signalling cascade and ECM proteins were evaluated by real-time polymerase chain reaction (TRT–PCR) and immunofluorescence microscopy. Animals with remnant kidneys developed hypertension, which was not relieved with paclitaxel treatment. However, paclitaxel treatment resulted in dampening the proteinuric response, reduction in serum BUN, creatinine levels and urine protein : creatinine ratio and normalization of creatinine clearance. These effects were accompanied by the inhibition of Smad2/3 activation, attenuation of renal fibrosis and normalization of integrin-linked kinase (ILK), COL(I)A1, COL(IV)A2 and α-SMA expression. Also, paclitaxel down-regulated the expression of miR-192, miR-217 and miR -377, while miR-15 was up-regulated in the remnant kidney. In vitro, in tubular epithelial cells (NRK-52E), paclitaxel also inhibited TGF-β1-induced Smad2/3 activation and normalized ILK, COL(I)A1, COL(IV)A2 and α-SMA expression. Furthermore, ChIP analyses indicated that Taxol suppressed Smad3-mediated miR-192 transcriptional activity. Over-expression of miR-192 in NRK-52E mimicked the changes seen in the remnant kidney, while inclusion of miR-192 inhibitor in the culture medium blocked TGF-β1-induced COL(I)A1 and COL(IV)A2 expression, while ILK and α-SMA were unaffected. These data suggest that low-dose paclitaxel ameliorates renal fibrosis via modulating miR-192 pathobiology and TGF-β/Smad signalling. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Characterization of an experimental model of progressive renal disease in rats

“…These models have the disadvantage that it is not always possible to establish the exact dose to produce a specific degree of renal insufficiency, in addition to the possible occurrence of side effects that may compromise the experimental model. On the other hand, the surgical procedures, characterized by the removal of portions of the renal tissue, have been widely used because they have a high survival rate and a low cost, do not have side effects and can be used to produce progressive levels of renal failure, as shown in this study [11][12][13][14][15][16] .…”

“…in creatinine clearance in rats and mice four weeks after 5/6 nephrectomy 12,13,21 . Under our experimental conditions, the creatinine clearance reduction in the group of severe uremia was of 87%.…”

“…The discrepancy of these results could be explained by the use of differente species and/or lineages of animals and due to differences in surgical protocols of renal tissue removal [11][12][13][14] .…”

“…These models are based on the use of chemical substances [8][9][10] or surgical produres that considerably reduce the renal tissue mass [11][12][13][14][15][16] . The pharmacologic models consist on the use of substance with nephrotoxic potential, which produce injuries to the renal tissue through different molecular mechanisms [8][9][10] .…”

“…This last model was originally described by Ormrod and Miller in 1980 11 , when the authors showed that surgical removal of a significant portion of the rat kidneys causes adaptive changes in remnant nephrons capable of leading to increase of glomerular filtration rate and urinary output. The subtotal nephrectomy model, which consists of unilateral nephrectomy associated to the removal of 5/6 of the remaining kidney, has been widely used as model of ESRD, defined as when the animal has 10% or less of its kidney function [11][12][13][14][15] . However, studies that followed the original description of Ormrod and Miller have not characterized the parameters of renal function and the histological changes resulting from CKD in this experimental of model of nephrectomy in different levels.…”