Oroxylin A ameliorates AKI-to-CKD transition through maintaining PPARα-BNIP3 signaling-mediated mitochondrial homeostasis

Background: Acute kidney injury (AKI) occurs in approximately 7–18% of all hospitalizations, but there are currently no effective drug therapy for preventing AKI or delaying its progression to chronic kidney disease (CKD). Recent studies have shown that Scutellaria baicalensis, a traditional Chinese herb, could attenuate cisplatin-induced AKI, although the mechanism remains elusive. Further, it is unknown whether its major active component, Oroxylin A (OA), can alleviate kidney injury. Methods: The therapeutic effect of OA was evaluated by using ischemia-reperfusion (IR) and cisplatin mediated-AKI mice and HK-2 cells under hypoxia-reoxygenation (HR) conditions. HE staining, transmission electron microscopy, flow cytometry, immunofluorescence, qPCR, Western blot, PPARα inhibitor, BNIP3 siRNA and ChIP assay were used to explore the role and mechanism of OA in AKI. Results: OA ameliorated tubular damage and dramatically decreased serum creatinine (Scr) and urea nitrogen (BUN), and the expressions of renal injury markers (Kim-1, Ngal) in AKI mice induced by both IR injury and cisplatin, as well as attenuating AKI-to-CKD transition. In vitro experiments showed that OA alleviated HR-induced mitochondrial homeostasis imbalance in renal tubular epithelial cells. Mechanistically, OA dose-dependently induced the expression of Bcl-2/adenovirus E1B 19-kDa interacting protein (BNIP3), while knockdown of BNIP3 expression reversed the protection of OA against HR-mediated mitochondrial injury. Network pharmacological analysis and experimental validation suggested that OA enhanced BNIP3 expression via upregulating the expression of peroxisome proliferator activated receptor alpha (PPARα), which induced the transcription of BNIP3 via directly binding to its promoter region. Both in vitro and in vivo experiments confirmed that the renoprotective effect of OA was dramatically reduced by GW6471, a PPARα antagonist. Conclusion: Our findings revealed that OA ameliorates AKI-to-CKD transition by maintaining mitochondrial homeostasis through inducing PPARα-BNIP3 signaling pathway, indicating that OA may serve as a candidate therapeutic strategy for alleviating AKI and CKD.


IRI and Cisplatin induced mouse models
Male C57BL/6J mice (8-week-old) were purchased from Chongqing Tengxin Bioscience (Chongqing, China). The construction of IRI-induced AKI mouse model was previously described (Hu et al., 2017). Brifely, mice were placed under general anesthesia for laparotomy, and their bilateral renal infarcts were clipped for 30 minutes by microaneurysm clips, then the mice were fed for another 24 hours for the construction of IRI-induced AKI model. To construct AKI-to-CKD model, bilateral renal ischemia of 35 minutes, followed by reperfusion for 2 weeks was used. To construct cisplatin-induced AKI model, 30 mg/kg of cisplatin was intraperitoneally injected into mice for 3 days as described in previous studies (Li et al., 2018).
To estimate the therapeutic effect of OA, mice were injected with control or OA (20 mg/kg) by tail vein after IRI surgery (Hu et al., 2017) or intraperitoneal cisplatin injection (Li et al., 2018). As reported, the kidney tissues and blood samples were collected (Hu et al., 2017, Li et al., 2018. Serum creatinine (Scr) and blood urea nitrogen (BUN) were determined by using assay kits (Nanjing Jiancheng Bioengineering Institute, China).

Cell Culture
Human proximal tubular epithelial cells (HK-2) were bought from the American Tissue Culture Collection (ATCC, Manassas, VA, USA) and cultured in F12 medium containing 10% fetal bovine serum (Mediatech, Manassas, VA, USA).

RNA Extraction and quantitative PCR (qPCR)
Total RNA was extracted from the mouse kidneys and HK-2 cells according to the manufacturers' instruction with an RNA extraction kit (Beyotime Biotechnology, Shanghai, China), which was reversely transcribed into cDNA with RT Master Mix for qPCR kit (MedChemExpress, Monmouth Junction, NJ, USA). qPCR was carried out by using a SYBR Green qPCR kit (MedChemExpress). The specific primers were listed in Supplementary Table 1 and 2.

Mitochondrial DNA (mtDNA) Copy Number Detection
Using the DNA of HK-2 cells as a template, the mtDNA copy number was detected by qPCR. The target gene mitochondria-encoded cytochrome c oxidase 2 (MT-CO2) and the internal reference glycerol-3-phosphate dehydrogenase (GAPDH) were detected respectively. The specific primers were listed in Supplementary Table 2.

Western Blot
Protein extraction and western blot analysis were performed as previously described . The signals were detected by an enhanced

Cell Viability Assay
After OA treatment of HK-2 cells for 48 hours, Cell Counting Kit-8 (CCK-8) solution (Beyotime Biotechnology, C0037) was added, co-incubating at 37 °C for another 1 hour, and the cell viability was analyzed by using Microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Apoptosis Analysis
After the treatment for indicated times, HK-2 cell solutions were harvested for staining with Annexin V-FITC/PI for 15 minutes using an Apoptosis Assay Kit (Beijing Labgic Technology, China), and apoptosis was measured by flow cytometry (Gallios, BD Biosciences).

ATP Measurement
The level of ATP was detected according to the manufacturer's instructions with an enhanced ATP assay kit (Beyotime Biotechnology, S0027).

Transfection of BNIP3 siRNA
To verify whether OA maintained mitochondrial homeostasis by inducing BNIP3 expression, the constructed BNIP3 siRNAs (Biomics Biotechnology, Chongqing, China) were transfected into HK-2 cells by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The BNIP3 siRNA and Negative control siRNA were listed in Supplementary Table 3.

Chromatin immunoprecipitation (ChIP) assay
The procedure for the CHIP experiment was described in our previous study . Briefly, the sonicated DNA was immunoprecipitated with 2 μg PPARα antibody, and then amplified by PCR and qPCR, whose primers were listed in Supplementary Table 4.

Statistical Analysis
All data were presented as mean ± SEM. Unpaired t test or one-way analysis of variance (ANOVA) with Tukey's test were used for statistical analysis. Statistically significant was defined as P < 0.05.  . (A, B) qPCR analysis of the expressions of oxidative phosphorylation and fatty acid oxidation-related genes in kidney tissues from sham and AKI mice treated with control or OA (n = 8). (C) Representative images of oil red O staining in kidney tissues from sham and AKI mice treated with control or OA. Scale bar, 50 μm. Data are presented as means ± SEM. ns: no significance. *P < 0.05, **P < 0.01, ***P < 0.001.