Inhibiting ERK Activation with CI-1040 Leads to Compensatory Upregulation of Alternate MAPKs and Plasminogen Activator Inhibitor-1 following Subtotal Nephrectomy with No Impact on Kidney Fibrosis

Abstract

Extracellular-signal regulated kinase (ERK) activation by MEK plays a key role in many of the cellular processes that underlie progressive kidney fibrosis including cell proliferation, apoptosis and transforming growth factor β1-mediated epithelial to mesenchymal transition. We therefore assessed the therapeutic impact of ERK1/2 inhibition using a MEK inhibitor in the rat 5/6 subtotal nephrectomy (SNx) model of kidney fibrosis. There was a twentyfold upregulation in phospho-ERK1/2 expression in the kidney after SNx in Male Wistar rats. Rats undergoing SNx became hypertensive, proteinuric and developed progressive kidney failure with reduced creatinine clearance. Treatment with the MEK inhibitor, CI-1040 abolished phospho- ERK1/2 expression in kidney tissue and prevented phospho-ERK1/2 expression in peripheral lymphocytes during the entire course of therapy. CI-1040 had no impact on creatinine clearance, proteinuria, glomerular and tubular fibrosis, and α-smooth muscle actin expression. However, inhibition of ERK1/2 activation led to significant compensatory upregulation of the MAP kinases, p38 and JNK in kidney tissue. CI-1040 also increased the expression of plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasmin-dependent matrix metalloproteinases. Thus inhibition of ERK1/2 activation has no therapeutic effect on kidney fibrosis in SNx possibly due to increased compensatory activation of the p38 and JNK signalling pathways with subsequent upregulation of PAI-1.

Fig 1. CI-1040 inhibits pERK1/2 activation and proliferation in rat fibroblasts.

NRK49F cells were serum starved overnight together with increasing concentrations of the MEK inhibitor CI-1040 prior to stimulation with 10% foetal bovine serum. pERK1/2 expression was assessed by western blotting with calnexin as a loading control (1a). CI-1040 treatment leads to a dose-dependent reduction in pERK1/2 expression as a percentage of control (n = 3). Cell proliferation as assessed by BrdU ELISA (1b) shows CI-1040 at doses between 100nM and 10,000nM has no significant effect on cell proliferation (closed circles). Viability assays (1b, closed triangles) determined CI-1040 was cytotoxic at doses higher than 10,00nM (assay performed 3 times in triplicate. V to refers vehicle only. + refers to FBS-stimulated cells and–refers to non-stimulated cells.

Fig 2. Phospho-ERK1/2 expression is increased after SNx and inhibited by CI-1040.

SNx was performed and western blotting demonstrated (a) pERK1/2 expression in homogenates of remnant kidneys was significantly increased at days 5 and 30 with some decline at day 90 (n = 4–5 for each time point). * p<0.05 compared to sham- operated controls. (b) 5 days after SNx, CI-1040 60mg/kg/day inhibited pERK1/2 expression in remnant kidneys (n = 5, * p<0.05 compared to SNx controls) and in (c) PMA- stimulated (+) lymphocytes extracted from pooled blood samples. There was no pERK1/2 expression in unstimulated (-) lymphocytes. Densitometry values were corrected for protein loading using calnexin and total ERK1/2.

Fig 3. CI-1040 has no effect on blood pressure, kidney function and albuminuria after SNx.

CI-1040 does not improve kidney function in the SNx rat; systolic blood pressure (SBP) (a), urinary albumin excretion (b), serum creatinine (c) and creatinine clearance (d) after 18 weeks treatment with either CI-1040 60mg/kg/d (closed triangles) or vehicle (closed circles) (n = 11 per group). Vertical bars indicate ± SEM, * p<0.05, ** p<0.001, *** p<0.0001 vs sham controls (open circles).

Fig 4. CI-1040 inhibits both renal and systemic pERK1/2 expression.

CI-1040 abolished pERK1/2 expression in renal homogenates following SNx (a). Vertical bars indicate ± SEM, *** p<0.0001 vs sham controls. Pharmacological exposure was demonstrated between doses systemically by inhibition of pERK1/2 expression from PMA-stimulated (+) lymphocytes. There is no pERK1/2 expression in unstimulated (-) lymphocytes (b). Lymphocytes were harvested prior to dosing (trough) and 30 minutes after dosing (peak). Serial harvesting of lymphocytes every 14 days demonstrated complete inhibition of pERK1/2 expression in PMA stimulated (+) lymphocytes throughout the time course of the experiment Example blots of peak lymphocyte activity at days 28, 56 and 84 (c). D = day.

Fig 5. Glomerulosclerosis and tubulointerstitial fibrosis after treatment with CI-1040.

Representative sections of glomeruli (400x) from terminal kidney tissue (131 days) stained with Masson’s trichrome obtained from (a) sham (c) SNx and (e) SNx plus CI-1040 60mg/kg/day rats. Group data are quantified in (g). Representative sections of the tubulointerstitium (200x) from terminal kidney tissue (131 days) stained with Masson’s trichrome obtained from (b) sham (d) SNx and (f) SNx plus CI-1040 60mg/kg/day rats. Group data are quantified in (h). Bars represent mean ± SEM, n = 7–11 per group* p<0.05, ** p<0.001 vs sham controls.

Fig 6. Myofibroblast number after treatment with CI-1040.

Myofibroblast number as indicated by α-SMA staining. Representative sections of glomeruli (400x) from terminal kidney tissue (90 day) stained with α-SMA obtained from (a) sham (c) SNx and (e) SNx plus CI-1040 60mg/kg/day rats. Group data are quantified in (g). Representative sections of the tubulointerstitium (200x) from terminal kidney tissue (90 day) stained with α-SMA obtained from (b) sham (d) SNx and (f) SNx plus CI-1040 60mg/kg/day rats. Group data are quantified in (h). Bars represent mean ± SEM, n = 7–11 per group. * p<0.05, *** p<0.001 vs sham controls.

Fig 7. CI-1040 inhibits phospho-ERK1/2 expression after SNx and upregulates p38, cJun and PAI-1.

(a) terminal kidney homogenates were western blotted for pERK1/2, phospho-p38 (p-p38), phospho-cJun (p-c-Jun) and PAI-1 with calnexin and total ERK1/2 acting as loading controls. (b) Densitometry values plotted with data representing mean +/- SEM, n = 6–11 per group. Statistical significance determined by one-way ANOVA. ***p<0.0001.