Analysis on promotive effect of rocking culture on keratinocyte differentiation in 3-dimensional reconstitution human epidermis.

A 3-dimensional culture system of keratinocytes achieves cornification as a terminal differentiation that can mimic the formation of stratified epidermis. At the onset of keratinocyte differentiation, air-exposure treatment is essential for promotion. We have previously reported that the stimulation of differentiation is accompanied by downregulation of the transcriptional activity of the hypoxia-inducible factor (HIF) and also found that rocking treatment of cultured keratinocytes in the submerged condition restored their differentiation. A comparative study of cultured keratinocytes with and without rocking was then carried out to investigate the characteristics of the recovered differentiation by morphological and biochemical analyses. In addition, transcriptome analysis revealed the expected similar pattern between air-exposed and rocking cultures, including HIF-regulating transcripts. Furthermore, the promotive effect of rocking treatment was impaired under hypoxic culture conditions (1% O2). We showed that the restored promotion of differentiation by rocking culture is mainly due to the abrogation of transcriptional events by hypoxia.

Novel function of transglutaminase 2 in extracellular histone-induced acute lung injury.

Extracellular histones induce endothelial damage, resulting in lung haemorrhage; however, the underlying mechanism remains unclear. Factor XIII, as a Ca2+-dependent cross-linking enzyme in blood, mediates fibrin deposition. As another isozyme, transglutaminase 2 (TG2) has a catalytic activity distributing in most tissues. Herein, we investigated whether TG2 promotes fibrin deposition and mediates the adhesion of platelets to ECs in histone-induced acute lung injury (ALI). We evaluated the lung histology and the adhesion of platelets to endothelial cells (ECs) after injecting histones to wild-type (WT) C57BL/6J and TG2 knockout (TG2-/-) mice, and administered a TG2 inhibitor (NC9) to WT mice. Pulmonary haemorrhage was more severe in TG2-/- mice than that in WT mice. The area of fibrin deposition and the proportion of CD41+CD31+ cells were lower in TG2-/- mice than in WT mice. Pre-treatment of NC9 decreased the area of fibrin deposition and the proportion of CD41+CD31+ cells in WT mice. These results suggest that TG2 prevents from pulmonary haemorrhage in ALI by promoting the adhesion of platelets to ECs and the fibrin deposition.

Development of peptide-based biosensors for detecting cross-linking and deamidation activities of transglutaminases.

Transglutaminases (TGs) are a protein family that catalyzes isopeptide bond formation between glutamine and lysine residues of various proteins. There are eight TG isozymes in humans, and each is involved in diverse biological phenomena due to their characteristic distribution. Abnormal activity of TG1 and TG2, which are major TG isozymes, is believed to cause various diseases, such as ichthyosis and celiac disease. To elucidate TGs' mechanisms of action and develop new therapeutic strategies, it is essential to develop bioprobes that can specifically examine the activity of each TG isozyme, which has not been sufficiently studied. We previously have identified several substrate peptide sequences containing Gln residues for each isozyme and developed a method to detect isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. We prepared the fluorescein isothiocyanate (FITC)-labeled Gln substrate peptide (FITC-K5 and FITC-T26) and Rhodamine B-labeled Lys substrate peptide (RhoB-Kpep). Each TG reaction specifically cross-linked these probe pairs, and the proximity of FITC and Rhodamine B significantly decreased the fluorescence intensity of FITC depending on the concentration and reaction time of each TG. In this study, we developed a peptide-based biosensor that quickly and easily measures TG isozyme-specific activity. This probe is expected to be helpful in elucidating TG's physiological and pathological functions and in developing compounds that modulate TG activity.

Inhibition of Transglutaminase 2 Reduces Peritoneal Injury in a Chlorhexidine-Induced Peritoneal Fibrosis Model.

Long-term peritoneal dialysis (PD) is often associated with peritoneal dysfunction leading to withdrawal from PD. The characteristic pathologic features of peritoneal dysfunction are widely attributed to peritoneal fibrosis and angiogenesis. The detailed mechanisms remain unclear, and treatment targets in clinical settings have yet to be identified. We investigated transglutaminase 2 (TG2) as a possible novel therapeutic target for peritoneal injury. TG2 and fibrosis, inflammation, and angiogenesis were investigated in a chlorhexidine gluconate (CG)-induced model of peritoneal inflammation and fibrosis, representing a noninfectious model of PD-related peritonitis. Transforming growth factor (TGF)-ß type I receptor (TGFßR-I) inhibitor and TG2-knockout mice were used for TGF-ß and TG2 inhibition studies, respectively. Double immunostaining was performed to identify cells expressing TG2 and endothelial-mesenchymal transition (EndMT). In the rat CG model of peritoneal fibrosis, in situ TG2 activity and protein expression increased during the development of peritoneal fibrosis, as well as increases in peritoneal thickness and numbers of blood vessels and macrophages. TGFßR-I inhibitor suppressed TG2 activity and protein expression, as well as peritoneal fibrosis and angiogenesis. TGF-ß1 expression, peritoneal fibrosis, and angiogenesis were suppressed in TG2-knockout mice. TG2 activity was detected by α-smooth muscle actin-positive myofibroblasts, CD31-positive endothelial cells, and ED-1-positive macrophages. CD31-positive endothelial cells in the CG model were α-smooth muscle actin-positive, vimentin-positive, and vascular endothelial-cadherin-negative, suggesting EndMT. In the CG model, EndMT was suppressed in TG2-knockout mice. TG2 was involved in the interactive regulation of TGF-ß. As inhibition of TG2 reduced peritoneal fibrosis, angiogenesis, and inflammation associated with TGF-ß and vascular endothelial growth factor-A suppression, TG2 may provide a new therapeutic target for ameliorating peritoneal injuries in PD.

Tissue transglutaminase exacerbates renal fibrosis via alternative activation of monocyte-derived macrophages.

Macrophages are important components in modulating homeostatic and inflammatory responses and are generally categorized into two broad but distinct subsets: classical activated (M1) and alternatively activated (M2) depending on the microenvironment. Fibrosis is a chronic inflammatory disease exacerbated by M2 macrophages, although the detailed mechanism by which M2 macrophage polarization is regulated remains unclear. These polarization mechanisms have little in common between mice and humans, making it difficult to adapt research results obtained in mice to human diseases. Tissue transglutaminase (TG2) is a known marker common to mouse and human M2 macrophages and is a multifunctional enzyme responsible for crosslinking reactions. Here we sought to identify the role of TG2 in macrophage polarization and fibrosis. In IL-4-treated macrophages derived from mouse bone marrow and human monocyte cells, the expression of TG2 was increased with enhancement of M2 macrophage markers, whereas knockout or inhibitor treatment of TG2 markedly suppressed M2 macrophage polarization. In the renal fibrosis model, accumulation of M2 macrophages in fibrotic kidney was significantly reduced in TG2 knockout or inhibitor-administrated mice, along with the resolution of fibrosis. Bone marrow transplantation using TG2-knockout mice revealed that TG2 is involved in M2 polarization of infiltrating macrophages derived from circulating monocytes and exacerbates renal fibrosis. Furthermore, the suppression of renal fibrosis in TG2-knockout mice was abolished by transplantation of wild-type bone marrow or by renal subcapsular injection of IL4-treated macrophages derived from bone marrow of wild-type, but not TG2 knockout. Transcriptome analysis of downstream targets involved in M2 macrophages polarization revealed that ALOX15 expression was enhanced by TG2 activation and promoted M2 macrophage polarization. Furthermore, the increase in the abundance of ALOX15-expressing macrophages in fibrotic kidney was dramatically suppressed in TG2-knockout mice. These findings demonstrated that TG2 activity exacerbates renal fibrosis by polarization of M2 macrophages from monocytes via ALOX15.

Involvement of hypoxia-inducible factor activity in inevitable air-exposure treatment upon differentiation in a three-dimensional keratinocyte culture.

Formation of the human skin epidermis can be reproduced by a three-dimensional (3D) keratinocyte culture system, in which air-exposure is inevitable upon initiation of differentiation. In the continuous submerged culture without air-exposure, even with a differentiation-compatible medium, several keratinocyte-specific proteins were not induced resulting in the formation of aberrant epidermal layers. To clarify the mechanism by which air-exposure promotes keratinocyte differentiation, we performed a comparative analysis on biological properties between submerged and air-liquid interphase culture systems. By transcriptomic analysis, hypoxia-inducible factor (HIF)-related genes appeared to significantly change in these cultured cells. In submerged culture, the transcriptional activity of HIF on its canonical response element was enhanced, while air-exposure treatment drastically reduced the transcriptional activity despite the high HIF protein level. Regulating HIF activity through reagents and genetic manipulation revealed that the reduced but retained HIF-transcriptional activity was essentially involved in differentiation. Furthermore, we showed, for the first time, that artificial supplementation of oxygen in the submerged culture system could restore keratinocyte differentiation as observed in the air-exposed culture. Thus, we mechanistically evaluated how HIF regulates the air-exposure-dependent differentiation of keratinocytes in a 3D culture system.

Biochemical characterization of medaka (Oryzias latipes) fibrinogen gamma and its gene disruption resulting in anemia as a model fish.

At the final stages of blood coagulation, fibrinogen is processed into insoluble fibrin by thrombin resulting in fibril-like structure formation. Via further cross-linking reactions between the fibrin gamma subunit by the catalytic action of blood transglutaminase (Factor XIII), this molecule gains further physical stability. Meanwhile, since fibrinogen is expressed in various cells and tissues, this molecule can exhibit other functions apart from its role in blood coagulation. To create a system studying on aberrant coagulation and investigate the physiological functions, using a model fish medaka (Oryzias latipes), we established gene-deficient mutants of fibrinogen gamma subunit protein in parallel with its biochemical analysis, such as tissue distribution pattern and substrate properties. By genetic deletion via genome editing, two distinct mutants displayed retardation of blood coagulation. The mutants showed lower hematocrit with aberrant erythrocyte maturation, which indicates that fibrin deficiency caused severe anemia, and also appeared as a model for investigation of the fibrin function.

Investigation of mouse amniotic fluid for stimulating ability of keratinocyte differentiation depending on the fetal stage.

During fetal development, the barrier function of the fetal skin is developed under specific conditions for epidermis formation. In keratinocyte differentiation, the well-orchestrated production and modification of various structural proteins are induced. We assessed the epidermal barrier function in different fetal stages by evaluating the enzymatic activity of cross-linking proteins, transglutaminases, and the permeation of fluorescence dye in the stained epidermal sections. During days 15.5-17.5 in gestation, the enzymatic activities in the epidermis appeared to increase significantly; meanwhile, dye permeation was substantially decreased, suggesting the formation of a protective barrier. For the fetal epidermis formation in the earlier stage, unclarified stimulating factors in the amniotic fluid (AF) are possible to promote barrier function by stimulating keratinocyte differentiation. Thus, we performed proteomic spectrometric (MS) analysis on the components in the AF at different fetal stages. Also, we investigated the promotive ability of the components using a cultured keratinocyte differentiation system. According to the MS analysis, the AF components appeared to exhibit stage-specific variations, where possible unique functions have been identified. We also found that adding the AF from each stage to the medium for cultured keratinocytes specifically enhanced the levels of the differentiation markers. These results provide information on the possible role of AF that contains regulatory factors on keratinocyte differentiation.

Role of Transglutaminase 2 in Cell Death, Survival, and Fibrosis.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme catalyzing the crosslinking between Gln and Lys residues and involved in various pathophysiological events. Besides this crosslinking activity, TG2 functions as a deamidase, GTPase, isopeptidase, adapter/scaffold, protein disulfide isomerase, and kinase. It also plays a role in the regulation of hypusination and serotonylation. Through these activities, TG2 is involved in cell growth, differentiation, cell death, inflammation, tissue repair, and fibrosis. Depending on the cell type and stimulus, TG2 changes its subcellular localization and biological activity, leading to cell death or survival. In normal unstressed cells, intracellular TG2 exhibits a GTP-bound closed conformation, exerting prosurvival functions. However, upon cell stimulation with Ca2+ or other factors, TG2 adopts a Ca2+-bound open conformation, demonstrating a transamidase activity involved in cell death or survival. These functional discrepancies of TG2 open form might be caused by its multifunctional nature, the existence of splicing variants, the cell type and stimulus, and the genetic backgrounds and variations of the mouse models used. TG2 is also involved in the phagocytosis of dead cells by macrophages and in fibrosis during tissue repair. Here, we summarize and discuss the multifunctional and controversial roles of TG2, focusing on cell death/survival and fibrosis.

Spatially Resolved Identification of Transglutaminase Substrates by Proteomics in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by the invariably progressive deposition of fibrotic tissue in the lungs and overall poor prognosis. TG2 (transglutaminase 2) is an enzyme that crosslinks glutamine and lysine residues and is involved in IPF pathogenesis. Despite the accumulating evidence implicating TG2 as a critical enzyme, the causative function and direct target of TG2 relating to this pathogenesis remain unelucidated. Here, we clarified the distributions of TG2 protein/activity and conducted quantitative proteomics analyses of possible substrates crosslinked by TG2 on unfixed lung sections in a mouse pulmonary fibrosis model. We identified 126 possible substrates as markedly TG2-dependently increased in fibrotic lung. Gene ontology analysis revealed that these identified proteins were mostly enriched in the lipid metabolic process, immune system process, and protein transport. In addition, these proteins were enriched in 21 pathways, including phagosome, lipid metabolism, several immune responses, and protein processing in endoplasmic reticulum. Furthermore, the network analyses screened out the six clusters and top 20 hub proteins with higher scores, which are related to endoplasmic reticulum stress and peroxisome proliferator-activated receptor signals. Several enriched pathways and categories were identified, some of which were the same terms based on transcription analysis in IPF. Our results provide novel pathological molecular networks driven by protein crosslinking via TG2, which can lead to the development of new therapeutic targets for IPF.

Thrombin-deficient mutant of medaka, a model fish, displays serious retardation in blood coagulation.

At the last stage of the blood coagulation cascade, thrombin plays a central role in the processing of fibrinogen for the polymerization and in the additional activation of Factor XIII for the stable cross-linking of fibrin. In addition, thrombin carries out possible multiple roles via processing or interaction with various functional proteins. Several studies conducted in order to elucidate additional physiological significance are ongoing. To clarify further significance of thrombin and to establish an associated disease model, we characterized the orthologue gene for medaka (Oryzias latipes), a research model fish. Tissue distribution of medaka prothrombin has been immunotechnically analyzed. Furthermore, thrombin-deficient medaka mutants were viably established by utilizing a genome-editing method. The established gene-deficient mutants exhibited retarded blood coagulation even in the heterozygous fish. Taking advantage of their ease of handling, this specific model is useful for further investigation in medical research areas on human coagulation diseases.

Gene disruption of medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2) causes movement retardation.

Transglutaminases are an enzyme family that catalyses protein cross-linking essential for several biological functions. In the previous studies, we characterized the orthologues of the mammalian transglutaminase family in medaka (Oryzias latipes), an established fish model. Among the human isozymes, tissue-type transglutaminase (TG2) has multiple functions that are involved in several biological phenomena. In this study, we established medaka mutants deficient for the orthologue of human TG2 using the CRISPR/Cas9 and transcription activator-like effector nucleases systems. Although apparent morphological changes in the phenotype were not observed, movement retardation was found in the mutant fish when evaluated by a tank-diving test. Furthermore, comparative immunohistochemistry analysis using in this fish model revealed that orthologue of human TG2 was expressed at the periventricular layer of the optic tectum. Our findings provide novel insight for the relationship between tissue-type transglutaminase and the nervous system and the associated behaviour.

Analysis of the expression of transglutaminases in the reconstructed human epidermis using a three-dimensional cell culture.

The skin epidermis functions as a barrier to various external stresses. In the outermost layer, the terminally differentiated keratinocytes result in cornification with a tough structure by formation of a cornified envelope beneath the plasma membrane. To complete the formation of the cornified envelope, several structural proteins are cross-linked via the catalytic action of transglutaminases (TG1, TG3, TG5, and TG6). The expression and activation of these enzymes are regulated in a tightly coordinated manner during keratinocyte differentiation. We here show the system detecting the activity of the TGases using specific glutamine-donor substrate peptides in a three-dimensional culture system of keratinocytes. In this review, we summarize the roles of the epidermal enzymes and introduce a detection method that will provide a system for evaluating the skin barrier function.

Identification and characterization of substrates crosslinked by transglutaminases in liver and kidney fibrosis.

The transglutaminase (TGase) family consists of eight isozymes that catalyze Ca2+-dependent crosslink formation between glutamine and lysine residues of proteins. In the pathogenesis of various chronic diseases, among the TGase isozymes, TG2 in particular is upregulated and contributes to a critical role in fibrosis development and progression via the stabilization of extracellular matrix proteins and activation of TGF-ß. Although TG2 has been considered a key enzyme in fibrosis, the causative role of TG2 and involvement of other isozymes remain unclear. We have recently developed a comprehensive analysis method targeting the isozyme-specific substrates of TGase in liver and kidney fibrosis. In this review article, we introduce a previously developed method for determining the activity and tissue distribution of TGase and for the detecting and identification of TGase substrates in an isozyme-specific manner. Using our comprehensive analysis method, we newly characterized the overlapping profile data regarding potential substrates of TG1 and TG2 that have been identified in liver and kidney fibrosis to date. Our results obtained by comparing the specificity and similarity of potential TGase substrates between different tissue fibrosis models provide a deeper understanding regarding the specific and common pathways in disease pathogenesis and progression.

Transglutaminase orthologues in medaka fish - biochemical characterization and establishment of gene-deficient mutants.

By genome analysis, seven homologous genes (orthologues) of human transglutaminases (TGases) have been identified in medaka fish (Oryzias latipes), some of which clearly corresponded to Factor XIII, TG1, and TG2. The enzymatically active-recombinant proteins for these medaka TGases have been successfully produced in bacteria or baculovirus-infected insect cell systems. Specific antibodies have been prepared and used in immunohistochemical analyses to reveal tissue distribution. Furthermore, gene-deficient medaka mutants for the genes encoding Factor XIII and TG1 have been established together with analysis of their phenotypes. Retarded cross-linking of fibrin and higher sensitivity to osmolality are observed when each gene is knocked-out. In this review, we summarize these biochemical features and the phenotypes of these gene-deficient fish.

Studies on differentiation-dependent expression and activity of distinct transglutaminases by specific substrate peptides using three-dimensional reconstituted epidermis.

During skin formation, particularly during differentiation of keratinocytes, unique post-translational modifications play a role in forming a proteinaceous supermolecule called the cornified envelope (CE), which is necessary for barrier function. Transglutaminases (TGs) are essential enzymes involved in the cross-linking of various keratinocyte structural proteins to complete CE formation. The TG family consists of eight isozymes, with two members, TG1 and TG3, located mainly in the epidermis. In an in vitro three-dimensional (3D) culture system, reconstruction of the epidermis allows cornification of the terminally differentiated keratinocytes. In this study, using isozyme-specific substrate peptides that enable detection of TG activity, we investigated the expression and the activation pattern of each isozyme during differentiation in this culture system. In the differentiating cells, the protein levels, enzymatic activities, as well as localization of TG1 and TG3 exhibited distinct patterns. Specific knockdown of these enzymes by siRNA revealed less cornification, suggesting that each TG contributes to the epidermal formation. In conclusion, we demonstrate the efficiency of the 3D system for studying differentiation-dependent expression and activity of distinct TGs by specific substrate peptides. ENZYME: Transglutaminase, EC2.3.2.13.

Detection and identification of potential transglutaminase 2 substrates in the mouse renal glomeruli.

The glomerulus primarily comprises mesangial cells, glomerular microvascular endothelial cells, and podocytes. IgA nephropathy is the most common primary glomerulonephritis worldwide and has a risk of progression to end-stage renal disease. IgA nephropathy is characterized by predominant IgA deposition in the glomerular mesangial area, where TG2 is significantly enhanced. Therefore, identification of glomerular TG2 substrates is the first step in elucidating the role of TG2 as a crosslinking enzyme during disease progression. To clarify potential glomerular TG2 substrates, and to establish a procedure for substrate identification, we attempted to identify those molecules using normal mouse glomeruli. Extracts from mouse glomerular and non-glomerular fractions were treated with our established biotin-labeled substrate peptide, which specifically crosslinks to the lysine-donor substrates depending on TG2 activity. Peptide-incorporated proteins were then purified using avidin resin and identified via mass spectrometry. In parallel, we performed the identification using corresponding samples from TG2 knockout mice. Consequently, potential TG2 substrates were separately identified in glomerular and non-glomerular fractions. They were mainly identified as novel TG2 substrates and partly include the well-known substrates. These results potentially provide novel insights into the mechanism underlying IgA nephropathy and may help elucidate the physiological functions of TG2.

Targeting transglutaminase 2 partially restores extracellular matrix structure but not alveolar architecture in experimental bronchopulmonary dysplasia.

The generation, maturation and remodelling of the extracellular matrix (ECM) are essential for the formation of alveoli during lung development. Alveoli formation is disturbed in preterm infants that develop bronchopulmonary dysplasia (BPD), where collagen fibres are malformed, and perturbations to lung ECM structures may underlie BPD pathogenesis. Malformed ECM structures might result from abnormal protein cross-linking, in part attributable to the increased expression and activity of transglutaminase 2 (TGM2) that have been noted in affected patient lungs, as well as in hyperoxia-based BPD animal models. The objective of the present study was to assess whether TGM2 plays a causal role in normal and aberrant lung alveolarization. Targeted deletion of Tgm2 in C57BL/6J mice increased septal thickness and reduced gas-exchange surface area in otherwise normally developing lungs. During aberrant lung alveolarization that occurred under hyperoxic conditions, collagen structures in Tgm2-/- mice were partially protected from the impact of hyperoxia, where normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance was restored; however, the lung alveolar architecture remained abnormal. Inhibition of transglutaminases (including TGM2) with cysteamine appreciably reduced transglutaminase activity in vivo, as assessed by Nε -(γ-l-glutamyl)-l-lysine abundance and TGM catalytic activity, and restored normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance under pathological conditions. Furthermore, a moderate improvement in alveoli size and gas-exchange surface density was noted in cysteamine-treated mouse lungs in which BPD was modelled. These data indicate that TGM2 plays a role in normal lung alveolarization, and contributes to the formation of aberrant ECM structures during disordered lung alveolarization.

Tschimganine and its derivatives extend the chronological life span of yeast via activation of the Sty1 pathway.

Most antiaging factors or life span extenders are associated with calorie restriction (CR). Very few of these factors function independently of, or additively with, CR. In this study, we focused on tschimganine, a compound that was reported to extend chronological life span (CLS). Although tschimganine led to the extension of CLS, it also inhibited yeast cell growth. We acquired a Schizosaccharomyces pombe mutant with a tolerance for tschimganine due to the gene crm1. The resulting Crm1 protein appears to export the stress-activated protein kinase Sty1 from the nucleus to the cytosol even under stressful conditions. Furthermore, we synthesized two derivative compounds of tschimganine, α-hibitakanine and ß-hibitakanine; these derivatives did not inhibit cell growth, as seen with tschimganine. α-hibitakanine extended the CLS, not only in S. pombe but also in Saccharomyces cerevisiae, indicating the possibility that life span regulation by tschimganine derivative may be conserved across various yeast species. We found that the longevity induced by tschimganine was dependent on the Sty1 pathway. Based on our results, we propose that tschimganine and its derivatives extend CLS by activating the Sty1 pathway in fission yeast, and CR extends CLS via two distinct pathways, one Sty1-dependent and the other Sty1-independent. These findings provide the potential for creating an additive life span extension effect when combined with CR, as well as a better understanding of the mechanism of CLS.

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

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-ß1. 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.