Intratubular epithelial-mesenchymal transition and tubular atrophy after kidney injury in mice

Abstract

Tubular atrophy is a common pathological feature of kidney fibrosis. Although fibroblasts play a predominant role in tissue fibrosis, the role of repairing tubular epithelia in tubular atrophy is unclear. We demonstrated the essential role of focal adhesion kinase (FAK)-mediated intratubular epithelial-mesenchymal transition (EMT) in the pathogenesis of tubular atrophy after severe ischemia-reperfusion injury (IRI). Actively proliferating tubular epithelia undergoing intratubular EMT were noted in the acute phase of severe IRI, resulting in tubular atrophy in the chronic phase, reflecting failed tubular repair. Furthermore, FAK was phosphorylated in the tubular epithelia in the acute phase of severe IRI, and its inhibition ameliorated both tubular atrophy and interstitial fibrosis in the chronic phase after injury. In vivo clonal analysis of single-labeled proximal tubular epithelial cells after IRI using proximal tubule reporter mice revealed substantial clonal expansion after IRI, reflecting active epithelial proliferation during repair. The majority of these proliferating epithelia were located in atrophic and non-functional tubules, and FAK inhibition was sufficient to prevent tubular atrophy. In vitro, TGFβ induced FAK phosphorylation and an EMT phenotype, which was also prevented by FAK inhibition. In an in vitro tubular epithelia gel contraction assay, TGFβ treatment accelerated gel contraction, which was suppressed by FAK inhibition. In conclusion, injury-induced intratubular EMT is closely related to tubular atrophy in a FAK-dependent manner.

Cumulative DNA damage by repeated low-dose cisplatin injection promotes the transition of acute to chronic kidney injury in mice

“…Interstitial fibrosis was quantified using the following scores: 0, 0%; 1, 1–10%; 2, 11–25%; 3, 26–50%; 4, 51–75%; and 5, 76–100%. Tubular injury was judged by tubular atrophy, tubular dilation, protein casts, necrotic cells, and brush border loss 31 .…”

“…Immunofluorescent staining was performed according to previously described method 31 . Sections were rehydrated and permeabilized with 0.5% Triton X-100 in PBS for 5 min.…”

“…Immunohistochemistry staining was performed according to previously described method 31 . After deparaffinization, the sections were placed in citrate-buffered solution (pH 6.0) and boiled for 5 min to retrieve antigens.…”

“…According to previously described method 31 , total RNA was extracted from the kidneys using TRIzol (Life Technologies, Inc., Carlsbad, CA) and Direct-zol™ RNA MiniPrep (Zymo Research Corporation., Irvine, CA). Two hundred nanograms of total RNA was reverse transcribed to synthesize cDNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan).…”

“…In general, after kidney injury and tubular cell death, neighboring dedifferentiated surviving cells play a key role by proliferative expansion with migration, which is followed by redifferentiation into mature tubular epithelia 28 – 31 . We hypothesized that cisplatin, an anticancer drug that generally causes DNA damage and cell death in rapidly proliferating cells, exacerbates DNA damage in regenerative proliferating renal proximal tubules after multiple rounds of cisplatin-induced injury and repair, thereby inducing extensive tubular damage and further maladaptive repair.…”