Isozyme-specific comprehensive characterization of transglutaminase-crosslinked substrates in kidney fibrosis

Hideki Tatsukawa,Ryosuke Wakita,Kiyotaka Hitomi,Risa Otsu,Yuji Tani

doi:10.1038/s41598-018-25674-4

Abstract

Chronic kidney disease is characterized by prolonged decline in renal function, excessive accumulation of ECM, and progressive tissue fibrosis. Transglutaminase (TG) is a crosslinking enzyme that catalyzes the formation of covalent bonds between glutamine and lysine residues, and is involved in the induction of renal fibrosis via the stabilization of ECM and the activation of TGF-β1. Despite the accumulating evidences indicating that TG2 is a key enzyme in fibrosis, genetic knockout of TG2 reduced by only 50% the elevated protein crosslinking and fibrous protein in renal fibrosis model, whereas treatment with TG inhibitor almost completely reduced these levels. Here, we also clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific crosslinked substrates for both TG1 and TG2 in fibrotic kidney. We found that TG1 activity was markedly enhanced in renal tubular epithelium and interstitial areas, whereas TG2 activity increased only in the extracellular space. In total, 47 and 67 possible candidates were identified as TG1 and TG2 substrates, respectively, only in fibrotic kidney. Among them, several possible substrates related to renal disease and fibrosis were identified. These findings provide novel insights into the mechanisms of renal fibrosis through the targeting of isozyme-specific TG substrates.

Highlights

Chronic kidney disease (CKD), including diabetic nephropathy and glomerulonephritis, is characterized by tubulointerstitial fibrosis and glomerulosclerosis that result from progressive remodeling processes, including excessive accumulation of extracellular matrix proteins (ECM), fibroblast proliferation and tubular atrophy[1]
We evaluated the enhanced activity of each TG isozyme, and globally identified the Lys-donor substrates for each TG isozyme and the Gln-donor substrates for the entire TG family during the induction of renal fibrosis
Evaluation of fibrotic markers in renal fibrosis induced by ureteral obstruction (UUO) surgery

Summary

Introduction

Chronic kidney disease (CKD), including diabetic nephropathy and glomerulonephritis, is characterized by tubulointerstitial fibrosis and glomerulosclerosis that result from progressive remodeling processes, including excessive accumulation of extracellular matrix proteins (ECM), fibroblast proliferation and tubular atrophy[1]. After 5/6-nephrectomy in rat[15] These results were evaluated in different animal and experimental model for fibrosis induction, TG inhibition rather than its genomic deficiency suggested an effective action for the prevention of fibrosis. These findings indicate that isozymes other than TG2 might have possible functions in fibrosis induction. Previous studies have shown that TG1 is mainly involved in skin formation, contributing to the barrier function of the outermost layers via crosslinking of structural proteins in keratinocytes[12,22], whereas TG2 is widely distributed and plays multiple roles, including in apoptosis, signal transduction, matrix stabilization, wound healing, and angiogenesis[10]. We suggested that TG1 is involved in the functional modification of intracellular protein, whereas TG2 predominantly contributes to the stabilization of extracellular proteins in liver fibrosis[23]

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