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Abstract Kidney fibrosis causes irreversible structural damage in chronic kidney disease and is characterized by aberrant extracellular matrix (ECM) accumulation. Although glutamyl-prolyl-tRNA synthetase 1 (EPRS1) is a crucial enzyme involved in proline-rich protein synthesis, its role in kidney fibrosis remains unclear. The present study revealed that EPRS1 expression levels were increased in the fibrotic kidneys of patients and mice, especially in fibroblasts and proximal tubular epithelial cells, on the basis of single-cell analysis and immunostaining of fibrotic kidneys. Moreover, C57BL/6 EPRS1tm1b heterozygous knockout (Eprs1 +/−) and pharmacological EPRS1 inhibition with the first-in-class EPRS1 inhibitor DWN12088 protected against kidney fibrosis and dysfunction by preventing fibroblast activation and proximal tubular injury. Interestingly, in vitro assays demonstrated that EPRS1-mediated nontranslational pathways in addition to translational pathways under transforming growth factor β-treated conditions by phosphorylating SMAD family member 3 in fibroblasts and signal transducers and activators of transcription 3 in injured proximal tubules. EPRS1 knockdown and catalytic inhibition suppressed these pathways, preventing fibroblast activation, proliferation, and subsequent collagen production. Additionally, we revealed that EPRS1 caused mitochondrial damage in proximal tubules but that this damage was attenuated by EPRS1 inhibition. Our findings suggest that the EPRS1-mediated ECM accumulation induces kidney fibrosis via fibroblast activation and mitochondrial dysfunction. Therefore, targeting EPRS1 could be a potential therapeutic target for alleviating fibrotic injury in chronic kidney disease.

Abstract Amphiregulin (AREG) is a transmembrane glycoprotein recently implicated in kidney fibrosis. Previously, we reported that the AREG-targeting Self-Assembled-Micelle inhibitory RNA (SAMiRNA-AREG) alleviated fibrosis by stably silencing the AREG gene, and reduced the side effects of conventional siRNA treatment of pulmonary fibrosis. However, the therapeutic effect of SAMiRNA-AREG in renal fibrosis has not been studied until now. We used two animal models of renal fibrosis generated by a unilateral ureteral obstruction (UUO) and an adenine diet (AD) to investigate whether SAMiRNA-AREG inhibited renal fibrosis. To investigate the delivery of SAMiRNA-AREG to the kidney, Cy5-labeled SAMiRNA-AREG was injected into UUO- and AD-induced renal fibrosis models. In both kidney disease models, SAMiRNA-AREG was delivered primarily to the damaged kidney. We also confirmed the protective effect of SAMiRNA-AREG in renal fibrosis models. SAMiRNA-AREG markedly decreased the UUO- and AD-induced AREG mRNA expression. Furthermore, the mRNA expression of fibrosis markers, including α-smooth muscle actin, fibronectin, α1(I) collagen, and α1(III) collagen in the UUO and AD-induced kidneys, was diminished in the SAMiRNA-AREG-treated mice. The transcription of inflammatory markers (tumor necrosis factor-α and monocyte chemoattractant protein-1) and adhesion markers (vascular cell adhesion molecule 1 and intercellular adhesion molecule 1) was attenuated. The hematoxylin and eosin, Masson’s trichrome, and immunohistochemical staining results showed that SAMiRNA-AREG decreased renal fibrosis, AREG expression, and epidermal growth factor receptor (EGFR) phosphorylation in the UUO- and AD-induced models. Moreover, we studied the effects of SAMiRNA-AREG in response to TGF-β1 in mouse and human proximal tubule cells, and mouse fibroblasts. TGF-β1-induced extracellular matrix production and myofibroblast differentiation were attenuated by SAMiRNA-AREG. Finally, we confirmed that upregulated AREG in the UUO or AD models was mainly localized in the distal tubules. In conclusion, SAMiRNA-AREG represents a novel siRNA therapeutic for renal fibrosis by suppressing EGFR signals.

The anti-aging gene, klotho, has been identified as a multi-functional humoral factor and is implicated in multiple biological processes. However, the effects of klotho on podocyte injury in diabetic nephropathy are poorly understood. Thus, the current study aims to investigate the renoprotective effects of klotho against podocyte injury in diabetic nephropathy. We examined lipid accumulation and klotho expression in the kidneys of diabetic patients and animals. We stimulated cultured mouse podocytes with palmitate to induce lipotoxicity-mediated podocyte injury with or without recombinant klotho. Klotho level was decreased in podocytes of lipid-accumulated obese diabetic kidneys and palmitate-treated mouse podocytes. Palmitate-treated podocytes showed increased apoptosis, intracellular ROS, ER stress, inflammation, and fibrosis, and these were significantly attenuated by klotho administration. Klotho treatment restored palmitate-induced downregulation of the antioxidant molecules, Nrf2, Keap1, and SOD1. Klotho inhibited the phosphorylation of FOXO3a, promoted its nuclear translocation, and then upregulated MnSOD expression. In addition, klotho administration attenuated palmitate-induced cytoskeleton changes, decreased nephrin expression, and increased TRPC6 expression, eventually improving podocyte albumin permeability. These results suggest that klotho administration prevents palmitate-induced functional and morphological podocyte injuries, and this may indicate that klotho is a potential therapeutic agent for the treatment of podocyte injury in obese diabetic nephropathy.