Abstract: SA-PO076
Induction of Hnf-1β Transcription Factor Protects Against Epithelial Hypoxia During Renal Ischemia-Reperfusion Injury
Session Information
- AKI: Mechanisms - Primary Injury and Repair - II
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Ruan, Mengna, Changzheng Hospital, Second Military Medical University, Shanghai, China
- Hao, Jielu, Mayo Clinic, Rochester, Minnesota, United States
- Wang, Zhenhua, Changzheng Hospital, Second Military Medical University, Shanghai, China
- Chen, Sixiu, Changzheng Hospital, Shanghai, China
- Zhou, Jie, Shuguang Hospital, Shanghai, China
- Mei, Changlin, Changzheng Hospital, Shanghai, China
Background
Ischemia-reperfusion injury is the crucial cause of acute kidney injury (AKI) in clinical settings. Proximal tubular cells are highly sensitive to ischemic injury and the following repair capability. Hnf-1β transcription factor drives the normal nephrogenesis and epithelial homeostasis. In the present study, we investigated the role of Hnf-1β regulation against hypoxic damage of proximal tubular cells in renal ischemia-reperfusion injury.
Methods
Renal ischemia-reperfusion was induced by bilateral clamping renal pedicles, the clamps were released for reperfusion. Kidney function was measured by BUN and serum creatinine, pathological damage was evaluated by HE and TUNEL stain. In vitro, cells were treated with hypoxia (1% oxygen) to represent ischemic condition, to test the effect of Hnf-1β, the editing-proficient Cas9 cell line was generated before hypoxia. The expression of Hnf-1β was tested by western blot and IHC. Flow cytometry and Hoechst stain were used to detect apoptosis. To explore the further effect of GJB1, the down-regulated stable cell line was created, and the expression was tested by realtime PCR. ChIP analysis was used to confirm the binding of NF-κB and Hnf-1β.
Results
Using western blot, we identified expression of Hnf-1β are significantly increased in kidney after 30 minutes of bilateral renal ischemia and reperfusion 12h, while the IHC staining showed the signal mainly in cortex. In vitro, hypoxia can also induce Hnf-1β in rat kidney epithelial cells (RPTC) as early as 4h and lasted to 12h. Interestingly, CRISPR/Cas9-mediated Hnf-1β knockout cells exacerbated apoptosis induced by hypoxic condition and caspase activity, whereas overexpression of Hnf-1β revealed more resistant to apoptotic responses in hypoxic cells. We further indicated these protective effects of Hnf-1β were mediated by NF-κB, which were confirmed by ChIP analysis, and its specific inactivator TPCA-1. Moreover, we also show that gap junction gene GJB1 could serve as downstream target of Hnf-1β as evidenced inhibition of GJB1 rescued Hnf-1β anti-apoptotic effect.
Conclusion
The study indicates the protective role of Hnf-1β against ischemic/hypoxic conditions in kidney. Hnf-1β performs anti-apoptotic factor mediated by NF-κB, and the renal protective effect may carry out via regulating its downstream gene GJB1.