Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling.

Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-β is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the macrolide calcineurin inhibitor tacrolimus (FK-506) induced TGF-β-like effects, manifested by increased expression of NAD(P)H-oxidase 4 (Nox4), transgelin, tropomyosin 1, and procollagen α1(V) mRNA after three days. The macrolide mTOR inhibitor rapamycin had similar effects, while cyclosporine A did not induce fibrose-related genes. Concentration dependence curves were sigmoid, where mRNA expression was induced already at low nanomolar levels of tacrolimus, and reached saturation at 100-300 nM. The effects were independent of extracellular TGF-β as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-β receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a "leaky" TGF-β receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-β1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-β1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H2O2 concentration. Si-RNA mediated knock-down of Nox4 expression prevented up-regulation of procollagen α1(V) mRNA in tacrolimus-treated cells, but induced procollagen α1(V) expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-β is a key molecule in fibrosis, and the constant activation of aberrant receptor signaling by tacrolimus might contribute to the long-term development of interstitial kidney fibrosis in immunosuppressed patients. Nox4 levels possibly play a regulatory role in these processes

Induction of fibrosis-related genes in TK-173 fibroblasts by nanomolar levels of tacrolimus.

<p>Exposure to increasing concentrations of tacrolimus (0–1000 nM) induced mRNA expression in a concentration-dependent manner after three days for Nox4 (A), transgelin (B), tropomyosin-1 (C), procollagen α1(V) (D), and transforming growth factor β-1 (E). a-smooth muscle actin mRNA expression (F) was not affected. RT-qPCR results were standardized to 18S rRNA, linearized, and normalized to the control group (without tacrolimus). (*) indicates significance from untreated cells p≤0,05.</p

Time course of mRNA expression in TK-173 cells cells treated with 100 nM tacrolimus.

<p>Expression of mRNA for NAD(P)H-oxidase 4 (A), transgelin (B), procollagen α1(V) (C), and alpha-smooth muscle actin (D) in untreated cells (open columns), and cells treated with 100 nM tacrolimus (filled). Cells were switched to serum-free medium on day -1, and were then exposed to 100 nM tacrolimus starting at day 0. Cell samples were collected from day 0 to day 5. All results were normalized to day 0. Cells showed a significant response to tacrolimus after one day (Nox4 and transgelin) or two days (tropomyosin-1), respectively. Alpha-smooth muscle actin mRNA showed a slow up-regulation in both treated and untreated cells, most likely as a reaction to serum deprivation. (*) denotes significant (p≤0,05) difference between tacrolimus-treated and control cells at the same timepoint.</p

Effect of siRNA-mediated Nox-4 knock-down in human TK-173 fibroblasts.

<p>Cells were transfected with Nox4-targeted siRNA (siNOX4), or non-target siRNA (si(-)) as a control, resp., and exposed to 100 nM tacrolimus, or control conditions, for three days. Transfection with Nox4-targeted siRNA reduced Nox4 mRNA expression by 61% (untreated control), and 64% (100 nM tacrolimus), resp., compared to non-target siRNA transfected cells (A). Nox4 knock-down did not have significant effects on transgelin (B), tropomyosin-1 (C), and alpha-smooth muscle actin (D) mRNA expression, but induced a significant up-regulation of mRNA for procollagen α1(V) in untreated cells, and a down-regulation to control levels in tacrolimus-treated cells (E). Results were normalized to control-transfected, untreated cells. (*) denotes significant (p≤0,05) differences between control-transfected and siRNA-transfected cells.</p

Effects of TGF-β1 signaling blockade.

<p>TK-173 cells exposed to 100 nM tacrolimus (TAC), or 10 ng/ml TGF-β1 (TGF) for three days showed increased expression of Nox4 (A), transgelin (B), and tropomyosin-1 (C). Blockade of TGF signaling by TGF-β1 RI inhibitor LY364947 inhibited both the reactions to TGF-β1 and tacrolimus [p≤0,05 (*), and p≤0,001 (**), resp.]. In contrast, application of anti-TGF-β antibody to the medium left the reaction to tacrolimus unaffected (E), but prevented the effects of 5 ng/ml TGF-β1 on the expression of procollagen α1(V) (Col5a1), Nox4, transgelin (Tagln), and tropomyosin-1 (Tmp1) almost completely (D). Antibodies had no effect in unstimulated control cultures (F). All values were normalized to untreated control cultures.</p

Comparison of the immunosuppressants cyclosporine A, tacrolimus, and rapamycin.

<p>Both tacrolimus (TAC; 100 nM) and rapamycin (RAPA, 100 nM) induced mRNA for Nox4, transgelin, and tropomyosin-1 after three days, although with a slightly different pattern. Cyclosporine A (CSA; 1 µM) had no effect on mRNA levels. All values normalized to untreated control. (*) denotes significance from untreated cells p≤0,05.</p

SMAD2 phosphorylation, and NOX4 expression and activity.

<p>Basal SMAD2 phosphorylation in untreated cells (Ctrl) is increased after 1 h, 2,5 h, and 5 h exposure to 100 nM tacrolimus (Tac). Preincubation with the TGF-β1-RI kinase inhibitors LY364947, or SB-431542 before exposure to tacrolimus (LY+Tac, and SB+Tac, resp.) ablates phosphorylation of SMAD2. 10 ng/ml TGF-β1 (TGF) served as a positive control. pSMAD2 band appeared at approx. 55 kD (A). Western blot for NOX4 protein in TK-173 fibroblasts treated over three days with 100 nM tacrolimus (Tac), 10 ng/ml TGF-β1 (TGF), and untreated control cells (Ctrl). A specific Nox4 band at 60 kD is present after stimulation of the cells with tacrolimus or TGF-β1, but undetectable in control cells (B). Intracellular H₂O₂ levels in TK-173 cells, expressed as relative DCF (dichlorofluorescin) fluorescence, were increased by tacrolimus in a concentration-dependent manner, following a sigmoid curve (C). Co-application of the TGF-β1-RI kinase inhibitor LY364947 significantly reduced intracellular H₂O₂ concentrations to control levels in cells treated with 100 nM tacrolimus (TAC), or 10 ng/ml TGF-β1 (D). Results were normalized to untreated control cells.</p

Increased Renal Versican Expression Is Associated with Progression of Chronic Kidney Disease

<div><p>Novel prognostic markers for progression of kidney disease are needed to distinguish patients who might benefit from a more aggressive nephroprotective therapy. Expression of the proteoglycan versican was evaluated in renal transcriptomics profiles and in an independent set of 74 renal biopsies. Versican levels were correlated to histologic damage scores and to renal outcome, and versican expression and regulation was evaluated <em>in vitro</em>. In transcriptomics profiles of renal tissue versican was positively correlated with (i) histological parameters in kidney biopsies, (ii) progressive decline of renal function in proteinuric kidney diseases, and (iii) impaired renal function and histology scores in diabetic nephropathy. In an independent cohort of 74 biopsies of glomerular diseases renal RNA levels of versican isoforms V0 and V1, but not V2 and V3 correlated significantly with creatinine after a mean follow up time of 53 months. Versican isoforms V0 and V1 together with serum creatinine at time of biopsy and the degree of glomerulosclerosis predicted 20% and 24% of the variability of creatinine at follow up, which was significantly more than serum creatinine and histological parameters alone (16%). However, when patients with acute kidney failure at time of biopsy (n = 5) were excluded, the additive predictive value of versican V1 was only marginally higher (35%) than creatinine and glomerulosclerosis alone (34%). Versican isoforms V0 and V1 were primarily expressed <em>in vitro</em> in proximal tubule cells and in fibroblasts. The results in humans were confirmed in three rodent models of kidney disease, in which renal versican expression was significantly upregulated as compared to corresponding controls. These data show for the first time an association of renal versican isoform V0 and V1 expression with progressive renal disease.</p> </div