To investigate the role and possible mechanism of celastrol (Cel) in sodium oxalate (NaOx)-induced acute kidney injury (AKI) and crystal deposition in the kidney tissues in mice.

Male C57BL/6 mice (aged 8~12 weeks, weighing 22~24 g) were randomly divided into 3 groups. Saline group (control group, intraperitoneal injection with normal saline and drinking water freely), NaOx group (injured group, intraperitoneal injection of 75 mg/kg NaOx, and drinking water containing 50 μmol/L NaOx), and NaOx+Cel group (treatment group, intraperitoneal injection of 1 mg/kg Cel firstly and then 75 mg/kg NaOx in 24 h later, drinking water containing 50 μmol/L NaOx). All specimens were collected in 24 h after NaOx injection. HK-2 cells were randomly divided into 4 groups: Medium group (no treatment), NaOx group (500 μmol/L NaOx), NaOx+Cel group (400 nmol/L Cel pre-treatment for 2 h followed by 500 μmol/L NaOx treatment), and NaOx+Cel+BA group [8 μmol/L betulinic acid (BA, NF-κB agonist) after the interventions as the NaOx+Cel group]. Cells of each group were collected in 24 h after corresponding treatments. Von Koosa and cell adhesion assays were used to observe crystal deposition. HE staining was employed to observe renal histopathology and score the damage. CCK-8 assay was utilized to detect cell viability to obtain the optimal concentrations of NaOx and Cel. Serum urea and creatinine levels were detected. Immunohisotochemical assay was conducted to detect the expression of OPN, CD44, KIM-1, NGAL, p65, IL-1β, BAX, and Caspase-3, and Western blotting was performed for protein levels of OPN, CD44, KIM-1, p65, P-p65 and IL-1β.

The mice in the NaOx+Cel group showed reduced crystal deposition (P<0. 0001), attenuated renal tubular damage (P<0. 01), decreased serum urea and creatinine levels (P<0. 05), and declined expression levels of the renal adhesion molecules OPN and CD44, the kidney injury molecules KIM-1 and NGAL, the inflammation-associated molecules p65 and IL-1β, and the apoptosis related molecules BAX and Caspase-3when compared with the NaOx group (P<0. 05). In in vitro study, the NaOx+Cel group showed reduced crystal adhesion (P<0. 0001), decreased expression of the adhesion molecules OPN and CD44 (P<0. 05), downregulation of the inflammatory molecule IL-1β and P-p65/p65 ratio (P<0. 05), and down-regulation of the renal injury molecule KIM-1 (P<0. 05) when compared with the NaOx group. In the NaOx+Cel+BA group, crystal adhesion was significantly increased (P<0. 0001), the inflammatory molecule IL-1β and the ratio of P-p65/p65were increased (P<0. 05), and the kidney injury molecule KIM-1 was increased when compared with the NaOx+Cel group (P<0. 05).

Cel may reduce NaOx-induced crystal deposition and AKI by inhibiting NF-κB activation.

To investigate the effect of hairy and enhancer of split 5 (HES5) on transdifferentiation and apoptosis of renal tubular epithelial cells induced by TGF-β1 and its potential mechanism.

The differentially expressed genes in GSE66494 data were analyzed and screened. The mouse model of unilateral uretera obstruction (UUO) was established, and the expression level of HES5 was detected in the renal tissue. HK-2 cells were treated with 10 ng/mL TGF-β1 for 24 h to establish a tubular epithelial-mesenchymal transition (EMT) model, and then qRT-PCR and Western blotting were performed to detect the expression of HES5 at mRNA and protein levels. After HK-2 cells were transfected with the plasmid overexpressing HES5, the protein levels of fibronectin, collagen Ⅰ, vimentin, apoptosis markers Bax and Bcl2 were detected in 24 h later. Then, HK-2 cells were divided into Control group, siHES5 group, TGF-β1 group, and siHES5+TGF-β1 group. The protein level of fibrosis and apoptosis markers were measured in above groups with Western blotting. TUNEL staining and flow cytometry were employed to detect cell apoptosis. Western blotting was applied to determine the protein levels of AKT, p-AKT, PI3K and p-PI3K. HK-2 cells overexpressing HES5 were treated with PI3K inhibitor LY294002, and the expression of vimentin was detected.

The expression of HES5 was significantly up-regulated in both chronic kidney disease (CKD) and fibrotic kidneys of mice. Overexpression of HES5 promoted the synthesis of fibronectin, collagen Ⅰ, vimentin and Bax in HK-2 cells, and inhibited the expression of Bcl2 (P<0.05). HES5 knockdown not only down-regulated the expression of fibrosis markers, but also inhibited the apoptosis of HK-2 cells. Furthermore, HES5 knockdown inhibited the activation of PI3K/AKT signaling pathway induced by TGF-β1 in HK-2 cells (P<0.05). Inhibitors of the PI3K/AKT signaling pathway inhibitor attenuated the induction of HES5 on vimentin.

HES5 knockdown inhibits the transdifferentiation and apoptosis in TGF-β1-induced renal tubular epithelial cells, which may be related to the decreased activity of the PI3K/AKT signaling pathway.