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

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

Transglutaminase inhibition ameliorates experimental diabetic nephropathy

Diabetic nephropathy is characterized by excessive extracellular matrix accumulation resulting in renal scarring and end-stage renal disease. Previous studies have suggested that transglutaminase type 2, by formation of its protein crosslink product epsilon-(gamma-glutamyl)lysine, alters extracellular matrix homeostasis, causing basement membrane thickening and expansion of the mesangium and interstitium. To determine whether transglutaminase inhibition can slow the progression of chronic experimental diabetic nephropathy over an extended treatment period, the inhibitor NTU281 was given to uninephrectomized streptozotocin-induced diabetic rats for up to 8 months. Effective transglutaminase inhibition significantly reversed the increased serum creatinine and albuminuria in the diabetic rats. These improvements were accompanied by a fivefold decrease in glomerulosclerosis and a sixfold reduction in tubulointerstitial scarring. This was associated with reductions in collagen IV accumulation by 4 months, along with reductions in collagens I and III by 8 months. This inhibition also decreased the number of myofibroblasts, suggesting that tissue transglutaminase may play a role in myofibroblast transformation. Our study suggests that transglutaminase inhibition ameliorates the progression of experimental diabetic nephropathy and can be considered for clinical application

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

<p>(<b>a</b>) 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>b</b>) Densitometry values plotted with data representing mean +/- SEM, n = 6–11 per group. Statistical significance determined by one-way ANOVA. ***p<0.0001.</p

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

<p>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.</p

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

<p>CI-1040 does not improve kidney function in the SNx rat; systolic blood pressure (SBP) (<b>a</b>), urinary albumin excretion (<b>b</b>), serum creatinine (<b>c</b>) and creatinine clearance (<b>d</b>) 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).</p

An IGF-1 antagonist does not inhibit renal fibrosis in the rat following subtotal nephrectomy.

Demonstrates for the first time that although IGF-1 contributes to compensatory renal growth there is no evidence to suggest from an interventional study that it has a role as a mediator of renal fibrosis