ERK1/2 Activation Following Renal Injury Is Required For Kidney Injury Molecule‐1 Production

Abstract

BackgroundBecause substantial damage can occur before detection of acute kidney injury (AKI) new early predictor biomarkers are needed, such as kidney injury molecule‐1 (KIM‐1). KIM‐1 is a transmembrane glycoprotein that is expressed at very low levels in healthy kidney and is highly upregulated during injury. KIM‐1 is shed during injury and can be detected in blood and urine. While KIM‐1 has been studied in various models of AKI, the regulatory mechanisms leading to KIM‐1 upregulation remains limited. Here, we explored the role of ERK1/2 activation on KIM‐1 expression following toxicant exposure to proximal tubule cells and renal ischemia reperfusion injury (IRI) and LPS‐induced sepsis in mice.MethodsMouse kidney proximal tubule cells (TK) were treated with varying concentrations of hydroxyurea (HU), hydrogen peroxide (H2O2), and tert‐butyl‐hydroperoxide (TBHP) with and without pretreatment with the MEK1/2 inhibitor trametinib (10 nM). Trametinib (1mg/kg) was administered 1 hr before renal IRI to C57Bl/6 mice and were euthanized at 3 hr and 24 hr. TLR4 KO and WT mice underwent 3 hr renal IRI or septic AKI for 18 hr using LPS (10 mg/kg) injection. Signaling pathways were explored using RT‐qPCR and immunoblot analysis and kidney function was measured using BUN and serum creatinine.ResultsHU, H2O2, and TBHP exposure to TK cells for 24 hr caused ERK1/2 phosphorylation and upregulated KIM‐1 mRNA. Pretreatment with trametinib prevented KIM‐1 up‐regulation and ERK1/2 phosphorylation at 24 hr. At 3 hr post renal IRI KIM‐1 mRNA increased 4‐fold while pretreatment with trametinib resulted in a 2‐fold increase in KIM‐1 mRNA. Serum creatinine increased 4‐fold 3 hr after IRI and was attenuated by trametinib pretreatment. Cortical KIM‐1 mRNA increased 600‐fold 24 hr after IRI and was reduced 66% using trametinib in IRI. KIM‐1 protein was observed at 24 hr but not at 3 hr post IRI. Trametinib decreased KIM‐1 protein in the kidney 24 hr post IRI. TLR4 KO mice subjected to IRI exhibited increased KIM‐1 mRNA and ERK1/2 phosphorylation after 3 hr. In contrast, TLR4 KO mice exposed to LPS did not exhibit increased KIM‐1 mRNA or protein, nor activated ERK1/2 after 18 hr. WT mice treated with LPS had increased KIM‐1 mRNA and protein and ERK1/2 phosphorylation.ConclusionWe have linked pathophysiological ERK1/2 phosphorylation to the regulation of KIM‐1 within the kidney cortex. ERK1/2 phosphorylation and KIM‐1 mRNA increased following toxicant exposure in TK cells, IRI in mice, and LPS in mice, and trametinib attenuated these increases. LPS‐induced AKI required TLR4 for ERK1/2 activation and subsequent upregulation of KIM‐1. In contrast, IRI mice did not require TLR4 for ERK1/2 activation. These results demonstrate that ERK1/2 is a key initiator of renal injury.Support or Funding InformationGM084147 ‐ RGSF31DK105782 ‐ JBC