Molecular Mechanisms of Kidney Fibrosis

Veterans Health Administration, Decatur PA

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Abstract

Acute kidney injury (AKI) predisposes to chronic kidney disease (CKD) which often progresses to organ failure. One of the hallmarks of CKD is tubulointerstitial fibrosis characterized by extracellular matrix accumulation, tubular atrophy and inflammatory cell infiltration. The main cells targeted by AKI are proximal tubule epithelial cells (PTECs) that contribute to CKD by producing pro-inflammatory cytokines and extracellular matrix and by stimulating myofibroblast differentiation of surrounding fibroblasts, leading to interstitial fibrosis. The goal of this grant is to investigate the molecular mechanisms whereby PTECs control pro-inflammatory and pro-fibrotic signaling in order to devise more effective therapies to halt and prevent AKI and its progression to CKD. Although many pathways have been implicated in both initiation and progression to kidney fibrosis, we will focus on Recepteur d'Origine Nantai (RON) and transforming growth factor beta (TGF-β) receptor II (TβRII). Based on the evidence that TβRII contains 5 phosphorylatable tyrosines and some of these tyrosines control activation of TβRII-mediated profibrotic signaling, we started to investigate the mechanisms whereby tyrosine phosphorylation of TβRII is controlled. We identified RON as a major receptor tyrosine kinase able to phosphorylate the cytoplasmic domain of TβRII. We show that RON and TβRII are expressed on PTECs and RON activation leads to TβRII tyrosine phosphorylation and enhances TβRII-mediated pro-inflammatory (e.g. IL-18 secretion) and pro-fibrotic (e.g. SMAD3 activation) signaling. In addition, we show that treatment of PTECs with the RON ligand MST1 leads to tyrosine phosphorylation of pro-fibrotic STAT3 and secretion of TGF-β, a key determinant in myofibroblast differentiation and development of tubulointerstitial fibrosis. Importantly, mice treated with the RON inhibitor Crizotinib show reduced AKI-induced proximal tubule injury characterized by decreased levels of tyrosine phosphorylated RON and TβRII, macrophage infiltration, SMAD3 and STAT3 activation, and collagen production. Based on these results and the finding that increased tyrosine phosphorylation of RON and TβRII is detected in the kidneys of mice subjected to severe AKI that progresses to CKD, we propose that RON in PTECs contributes to kidney injury and fibrosis by promoting 1) tyrosine phosphorylation of TβRII thus enhancing TβRII-mediated production of pro-inflammatory cytokines (IL-18) and activation of pro-fibrotic molecules (SMAD3); and 2) TGF-β secretion in a STAT3 dependent manner thus inducing myofibroblast differentiation. The aims of this grant are designed to define the contribution of RON/STAT3 and RON/TβRII axes in kidney disease and to determine if their inhibition is beneficial in slowing/halting AKI and its progression to CKD. In Aim 1 we will study the role of RON-mediated phosphorylation of TβRII in AKI and AKI-to-CKD progression using a genetic and pharmacological approach. We will investigate the response of mice expressing a kinase dead RON (RONTK-/-) and RONTK-/- mice crossed to mice lacking TβRII in proximal tubules to severe AKI that progresses to CKD. We will determine whether IL-18 mediates RON/TβRII pro-inflammatory action by blocking IL18-mediated function with IL-18-binding protein. Finally, we will translate the relevance of these findings to a more clinically relevant setting by investigating the response to kidney injury in mice treated with a small molecule RON inhibitor. In Aim 2 we will determine the mechanisms whereby RON contributes to inflammation and fibrosis. We will use protein assays and in vitro cell cultures to determine the role of STAT3 in RON-mediated TGF-β production. We will also identify the key tyrosines on TβRII directly phosphorylated by RON and PTEC cultures to determine their role in regulating pro-inflammatory and pro-fibrotic signaling. Understanding how the RON/STAT3 and RON/TβRII axes regulate kidney injury and exploring the consequences of RON inhibition in reducing TGF-β-mediated pro-inflammatory and pro-fibrotic responses will offer a novel approach for the treatment and, ideally, prevention of CKD and ultimately end stage kidney disease.