Tacrolimus and Cyclosporinein vitro and in vivo Induce Osteopontin mRNA and Protein Expression in Renal Tissues

Abstract

The mechanism of immunosuppression-linked nephrotoxicity in organ transplantation remains to be solved. Expression of osteopontin (OPN), a multifunctional secreted glycoprotein, has been associated with various forms of renal injuries. In this study, using in vitro and in vivo models, we examined the effects of cyclosporine (CsA) and tacrolimus (TAC) on OPN mRNA and protein expression. We also examined if CsA- and TAC-induced OPN expression is dependent on transforming growth factor (TGF)-β expression. For in vivo experiments mice and rats were injected with CsA (25 mg/kg) and TAC (0.2 mg/kg). For in vitro experiments, human proximal tubular epithelial (PTE) cells were treated with CsA and TAC for 4 h. To study the in vivo effect of TGF-β on OPN mRNA, mice were injected with recombinant TGF-β protein (3 mg/kg). The expression of OPN was also studied in CsA-treated PTE cells with and without anti-TGF-β antibody. At the end of in vitro and in vivo treatments, RNA was isolated from kidney tissue and renal cells reverse transcribed to cDNA and amplified for OPN mRNA. Using immunochemistry and Western blot analysis OPN protein expression was also studied in vivo and in vitro, respectively. Both in vitro and in vivo treatment with CsA and TAC resulted in significantly increased OPN mRNA and protein expression. TGF-β treatment in vivo also resulted in a significantly increased OPN mRNA expression and anti-TGF-β antibody but not the control antibody in vivo in CsA-treated mice, and in vitro in CsA-treated PTE cells inhibited OPN mRNA expression. OPN may contribute to the CsA- and TAC-induced nephrotoxicity in organ transplant recipients and the increased OPN expression might be mediated by TGF-β.

Adverse Effects of Nicotine and Immunosuppression on Proximal Tubular Epithelial Cell Viability, Tissue Repair and Oxidative Stress Gene Expression

“…The relative mRNA level is presented as 2[(Ct/␤-actin Ϫ Ct/gene of interest)]), as described elsewhere. 25,26 The results are presented as normalized fold expression using ␤-actin as a reference gene and untreated cells as control.…”

“…25,26 The membranes were stripped with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific, Inc., Champaign, IL) and probed with anti-␤-actin antibody (Cell Signaling Technology, Inc., Danvers, MA).…”

“…The combinations of N with SRL (N ϩ S), MMF (N ϩ M) and SRL ϩ MMF (N ϩ S ϩ M) resulted in26.1 Ϯ 2.72-fold, 17.8 Ϯ 0.97-fold and 92.1 Ϯ 11.8-fold (p Ͻ 0.001) increases, respectively, compared with SRL alone (Figure 4A). Similarly, gp91 mRNA expression increased after treatment with N (16.45 Ϯ 1.35-fold, p Ͻ 0.003), SRL (16.58 Ϯ 2.23-fold, p Ͻ 0.002) and MMF (11.91 Ϯ 1.69-fold, p Ͻ 0.004).…”

Can Peptidomics Provide a Useful Approach for the Identification of Biomarkers of Toxicological Exposure or Effect?

“…The synthesis and secretion of transforming growth factor (TGF)-b by renal tubule epithelial cells appears to be central to this process [24][25][26], and thus measurement of TGF-b and its induced peptides/proteins may have a clinical application in monitoring kidney damage during CsA therapy. The pivotal role of TGF is illustrated by the effective suppression of cyclosporine A toxicity through the introduction of a neutralizing receptor, TGF-b 2R, into rats by gene transfer [24].…”

“…The pivotal role of TGF is illustrated by the effective suppression of cyclosporine A toxicity through the introduction of a neutralizing receptor, TGF-b 2R, into rats by gene transfer [24]. Once expressed after CsA exposure, TGF-b is reported to increase expression of osteopontin [26] and TGF-b-induced gene h3 [25] in a dose-dependent manner, where urinary betaig-h3 has been proposed as a sensitive urinary marker of nephrotoxicity. Betaig-h3 is a 72 kDa protein and, therefore, should be detectable by conventional proteomic techniques.…”