Activation of mTORC1 (mechanistic target of rapamycin complex 1) in renal tissue has been reported in chronic kidney disease (CKD)-induced renal fibrosis. However, the molecular mechanisms responsible for activating mTORC1 in CKD pathology are not well understood. The purpose of this study was to identify the uremic toxin involved in mTORC1-induced renal fibrosis. Among the seven protein-bound uremic toxins, only indoxyl sulfate (IS) caused significant activation of mTORC1 in human kidney 2 cells (HK-2 cells). This IS-induced mTORC1 activation was inhibited in the presence of an organic anion transporter inhibitor, a NADPH oxidase inhibitor, and an antioxidant. IS also induced epithelial–mesenchymal transition of tubular epithelial cells (HK-2 cells), differentiation of fibroblasts into myofibroblasts (NRK-49F cells), and inflammatory response of macrophages (THP-1 cells), which are associated with renal fibrosis, and these effects were inhibited in the presence of rapamycin (mTORC1 inhibitor). In in vivo experiments, IS overload was found to activate mTORC1 in the mouse kidney. The administration of AST-120 or rapamycin targeted to IS or mTORC1 ameliorated renal fibrosis in Adenine-induced CKD mice. The findings reported herein indicate that IS activates mTORC1, which then contributes to renal fibrosis. Therapeutic interventions targeting IS and mTORC1 could be effective against renal fibrosis in CKD.

Renal ischemia-reperfusion (IR)-induced tissue hypoxia causes impaired energy metabolism and oxidative stress. These conditions lead to tubular cell damage, which is a cause of acute kidney injury (AKI) and AKI to chronic kidney disease (CKD). Three key molecules, i.e., hypoxia-inducible factor-1α (HIF-1α), AMP-activated protein kinase (AMPK), and nuclear factor E2-related factor 2 (Nrf2), have the potential to protect tubular cells from these disorders. Although carbon monoxide (CO) can comprehensively induce these three molecules via the action of mitochondrial reactive oxygen species (mtROS), the issue of whether CO induces these molecules in tubular cells remains unclear. Herein, we report that CO-enriched red blood cells (CO-RBC) cell therapy, the inspiration for which is the in vivo CO delivery system, exerts a renoprotective effect on hypoxia-induced tubular cell damage via the upregulation of the above molecules. Experiments using a mitochondria-specific antioxidant provide evidence to show that CO-driven mtROS partially contributes to the upregulation of the aforementioned molecules in tubular cells. CO-RBC ameliorates the pathological conditions of IR-induced AKI model mice via activation of these molecules. CO-RBC also prevents renal fibrosis via the suppression of epithelial mesenchymal transition and transforming growth factor-β1 secretion in an IR-induced AKI to CKD model mice. In conclusion, our results confirm that the bioinspired CO delivery system prevents the pathological conditions of both AKI and AKI to CKD via the amelioration of hypoxia inducible tubular cell damage, thereby making it an effective cell therapy for treating the progression to CKD.

Fat atrophy and adipose tissue inflammation can cause the pathogenesis of metabolic symptoms in chronic kidney disease (CKD). During CKD, the serum levels of advanced oxidation protein products (AOPPs) are elevated. However, the relationship between fat atrophy/adipose tissue inflammation and AOPPs has remained unknown. The purpose of this study was to investigate the involvement of AOPPs, which are known as uremic toxins, in adipose tissue inflammation and to establish the underlying molecular mechanism. In vitro studies involved co-culturing mouse-derived adipocytes (differentiated 3T3-L1) and macrophages (RAW264.7). In vivo studies were performed using adenine-induced CKD mice and AOPP-overloaded mice. Fat atrophy, macrophage infiltration and increased AOPP activity in adipose tissue were identified in adenine-induced CKD mice. AOPPs induced MCP-1 expression in differentiated 3T3-L1 adipocytes via ROS production. However, AOPP-induced ROS production was suppressed by the presence of NADPH oxidase inhibitors and the scavengers of mitochondria-derived ROS. A co-culturing system showed AOPPs induced macrophage migration to adipocytes. AOPPs also up-regulated TNF-α expression by polarizing macrophages to an M1-type polarity, and then induced macrophage-mediated adipose inflammation. In vitro data was supported by experiments using AOPP-overloaded mice. AOPPs contribute to macrophage-mediated adipose inflammation and constitute a potential new therapeutic target for adipose inflammation associated with CKD.

Cisplatin-induced acute kidney injury (AKI) is an important factor that limits the clinical use of this drug for the treatment of malignancies. Oxidative stress and inflammation are considered to be the main causes of not only cisplatin-induced death of cancer cells but also cisplatin-induced AKI. Therefore, developing agents that exert antioxidant and anti-inflammatory effects without weakening the anti-tumor effects of cisplatin is highly desirable. Carbon monoxide (CO) has recently attracted interest due to its antioxidant, anti-inflammatory, and anti-tumor properties. Herein, we report that CO-loaded red blood cell (CO-RBC) exerts renoprotective effects on cisplatin-induced AKI. Cisplatin treatment was found to reduce cell viability in proximal tubular cells via oxidative stress and inflammation. Cisplatin-induced cytotoxicity, however, was suppressed by the CO-RBC treatment. The intraperitoneal administration of cisplatin caused an elevation in the blood urea nitrogen and serum creatinine levels. The administration of CO-RBC significantly suppressed these elevations. Furthermore, the administration of CO-RBC also reduced the deterioration of renal histology and tubular cell injury through its antioxidant and anti-inflammatory effects in cisplatin-induced AKI mice. Thus, our data suggest that CO-RBC has the potential to substantially prevent the onset of cisplatin-induced AKI, which, in turn, may improve the usefulness of cisplatin-based chemotherapy.